JP5204761B2 - Methods of treating cell proliferative disorders by using pyrimidinediamine compounds - Google Patents

Abstract

The present disclosure provides methods for the treatment of cell proliferative disorders by administration of a Syk kinase or Syk/Flt-3 kinase inhibitor. Cell proliferative disorders treatable by the methods include, hematopoietic neoplasms and virally associated tumors. The compounds are also directed to therapeutic or prophylactic inhibition of tumor metastasis.

Description

(Related application)
This application claims priority to US patent application Ser. No. 11 / 407,233, filed Apr. 18, 2006, the entire contents of which are hereby incorporated by reference.

(Technical field)
The present disclosure relates to methods and compositions for the treatment of cell proliferative disorders, the compositions comprising inhibitors that target kinase activity that affects the proliferative potential of the cells.

(background)
Unregulated cell growth is a hallmark of tumors and cancers and other cell proliferative disorders. The cellular processes that control cell division and proliferation are complex, with complex interactions between gene products that promote cell division and growth and gene products that block such processes. Positive regulators of growth and proliferation are generally described as pre-oncogenes, which are modified genes and the normal counterparts of these gene products known to promote tumor and cancer formation. Pre-oncogenes promote cell division and negatively regulate cell apoptosis. Uncoupling of the activity of these gene products from these normal regulatory states converts pre-oncogenes to oncogenes. Normal functions of pre-oncogenes include growth factors, growth factor receptors, cellular signaling molecules, and nuclear factors. Activation of pre-oncogenes to oncogene forms can occur in a variety of ways. This includes gene mutation, amplification, gene translocation, and viral activation.

  In contrast to pre-oncogenes, tumor suppressors generally have a negative effect on cell growth, promote cell apoptosis, inhibit cell cycle progression, and affect the likelihood of invasion and metastasis. give. In some instances, tumor suppressors can counteract the activity of oncogenes even in these modified forms. Upon loss or inhibition of tumor suppressor function, unregulated activity of pre-oncogenes or their corresponding oncogene forms results in cell transformation and carcinogenesis. Gene mutations or deletions, inhibitory transcription, increased degradation, or abnormalities in related proteins that work in concert with tumor suppressors can compromise tumor suppressor activity. Tumor suppressor genes act as recessive alleles so that cells that have normal alleles with mutant alleles still behave normally. Thus, loss of normal alleles, also called loss of heterozygosity (LOH), characterizes several types of abnormal cell growth and proliferation. Genomic instability resulting from disruption of oncogene activity and normal cell division control can increase the probability of LOH and thus the occurrence of a transformed phenotype by the oncogene.

  Treatment of cell proliferative disorders may target oncogenes and / or tumor suppressors that are affected in transformed cells. However, a loss of function, such as a disorder due to a tumor suppressor, is typically in a disorder resulting from altered acquisition of function, such as activation of an oncogene, when trying to treat the underlying molecular defect. More problematic than treating underlying molecular defects. Modification of cellular processes to give lost cell function is impractical in many cases. In this way, even for cell proliferative disorders resulting from loss of tumor suppressor activity, treatment typically leads to dysregulated molecules (eg, pre-oncogenes) that act as a result of lost tumor suppressor function. Directed. Although many molecular targets have been identified, such as non-receptor and receptor-based protein kinases, the complex nature of the cellular regulatory mechanisms that are effective in cell proliferation and growth is that other molecules that can be therapeutic targets It will indicate that it has not yet been identified. Some of these will be unknown, while others may not be associated with cell proliferative disorders.

  Thus, other cells that act oncogeneically in cell proliferative disorders, either as a result of alteration of its own activity or as a result of loss of cellular function that acts to regulate its activity It is desirable to identify the molecule. In identifying such molecules, compounds specifically directed to that cellular molecule can be identified and used to treat cell proliferative disorders, either independently or in combination with other known therapies be able to.

(Summary)
The present disclosure provides a method of treating a cell proliferative disorder by administering to a subject an amount of a Syk kinase inhibitor compound effective to treat the cell proliferative disorder. In some embodiments, the Syk kinase inhibitor is selective for Syk kinase, thereby specifically targeting aberrant Syk kinase activity present in a proliferative disorder. Any cell proliferative disorder in which Syk plays a role in some aspects of abnormal cell division or cell growth can be treated with these inhibitory compounds. In some embodiments, the cell proliferative disorders treatable with these inhibitory compounds are hematopoietic neoplasms, which are abnormal growth involving cells of the hematopoietic lineage. Hematopoietic neoplasms treatable with these Syk inhibitor compounds include, among other things, various bone marrow and lymphoid neoplasms such as chronic myelogenous leukemia, Burkitt lymphoma, and acute myeloid leukemia.

  In some embodiments, the Syk kinase inhibitor compound comprises a Syk / Flt-3 kinase inhibitor compound that can inhibit the activity of both Syk kinase and Flt-3 kinase. These compounds can be used to treat cell proliferative disorders associated with abnormal Flt-3 kinase activity. Various hematopoietic neoplasms with abnormal Flt-3 activity include, among others, acute myeloid leukemia, B-progenitor cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, myelodysplastic syndrome, and chronic Includes myeloid leukemia. These and other such disorders involving Flt-3 can be treated with Syk / Flt-3 inhibitory compounds, for example members of the 2,4-pyrimidinediamine compounds described herein.

  In other embodiments, these inhibitory compounds can be used to treat tumors mediated by viral genes that transform normal cells into tumor cells. In these embodiments, tumors whose viral oncogene activity disrupts Syk kinase function as part of its transformation mechanism can be targeted for therapy. Thus, in some embodiments, the virus-mediated tumor is associated with infection by a virus carrying a gene encoding a protein having an immunoreceptor tyrosine-based activation motif (ITAM). Usually, these conserved sequences regulate Syk kinase activity during the development and function of immune system cells (eg, B cells, T cells, neutrophils, etc.). However, permanent expression of viral proteins with ITAM sequences can result in aberrant Syk kinase activity and resulting tumor formation and / or maintenance. In various embodiments, the virus-mediated tumor may be associated with Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, human T-cell lymphotropic virus (HTLV-1), or mammary adenocarcinoma virus (MTV). . The presence of these viruses correlates with several cell proliferative disorders. This includes certain forms of Kaposi sarcoma, Burkitt lymphoma, Hodgkin lymphoma, adult T-cell leukemia, and breast cancer.

  In some embodiments, the disclosure provides a method of inhibiting tumor metastasis, comprising administering to a subject an amount of a Syk kinase inhibitor compound effective to inhibit tumor metastasis. Any tumor that has the potential to metastasize can be treated with these inhibitory compounds. In some embodiments, tumor metastasis is associated with integrin activity. They can act through Syk kinase in regulating cell adhesion properties. For example, it is observed in metastasis of tumor cells. Syk kinase inhibitors can be used to inhibit tumor metastasis by affecting cell signaling events mediated by integrins, such as β1, β2, and / or β3 integrins.

  In some embodiments, these inhibitory compounds can be used in combination with other cancer therapies. In some embodiments, the Syk inhibitor compound is used in combination with other chemotherapeutic agents. This includes inter alia antimetabolites, alkylating agents, coordination compounds, transcription inhibitors, topoisomerase inhibitors, DNA minor groove binding compounds, vinca alkyloids, antitumor antibiotics, hormones, and antitumor enzymes.

(Detailed description of the invention)
6.1 Treatment Methods The present disclosure provides methods for treating cell proliferative disorders by administering a Syk kinase or a Syk / Flt-3 kinase inhibitor compound. Syk kinase is one of two known members of the Syk family (Syk and ZAP-70) non-receptor tyrosine kinases. Syk is activated upon binding of its src homolog 2 (SH2) domain to an immunoreceptor tyrosine-based activation motif (ITAM). Syk kinase plays an essential role in lymphocyte development and immune cell activation and is best characterized for its role in B cell receptor signaling and Fc receptor mediated release of mast cell granules. Syk is ubiquitously expressed in hematopoietic cells, but is also expressed in other tissues such as breast epithelial cells and hepatocytes. Some believe in this field that Syk is a tumor suppressor and acts as a negative regulator of metastatic potential. Loss of Syk activity is suggested to be associated with the formation of invasive breast cancer, and chromosomal loss of the Syk gene has been shown in some types of lymph node metastasis of primary breast cancer. Thus, the use of Syk inhibitors for the treatment of cell proliferative disorders or as a treatment to reduce the likelihood of tumor cell metastasis would be contraindicated when Syk functions as a tumor suppressor. .

  Flt-3 is also a tyrosine kinase, but unlike Syk, it belongs to the tyrosine kinase receptor protein family. Flt-3 is a member of class III receptor tyrosine kinases, which are related by amino acid sequence and structural properties. Flt-3 is activated by binding to its cognate ligand Flt-3L. Flt-3 kinase is expressed in early bone marrow early hematopoietic stem cells and appears to function in the development of pluripotent stem cells and B cells by regulating the activity of various downstream cell targets. These include, among others, phospholipase C- (PLC), the p85 subunit of phosphatidylinositol 3′-kinase (PI3K), Shc, Shp-2, Ship, Grb2, Vav, Fyn kinase, Src kinase, Stat5 signaling protein, and Includes Erk. Although Flt-3 kinase is normally expressed in neural progenitor cells in the bone marrow, high levels of expression are also present in blood cell proliferative disorders such as acute myeloid leukemia (AML), B-progenitor cell acute lymph It is also observed within the scope of blastic leukemia, T-cell acute lymphoblastic leukemia, and chronic myeloid leukemia (CML) in lymphoblast transformation (eg, Rosnet et al., 1996, Leukemia 10: 238-248; Carow et al. , 1996, Blood 87: 1089-1096). The presence of Flt-3 mutations in blood cell proliferative disorders correlates with decreased remission rates, increased relapse rates, and generally lower overall survival (Rombouts et al., 2000, Leukemia 14: 675-683). Theide et al., 2002, Blood 99: 4326-4335; Frohling et al., 2002, Blood 100: 4372-4380).

  Although the industry suggests that Syk can act as a tumor suppressor, the present disclosure is based on the indication that Syk performs a function opposite to its hypothesized role. For example, forced expression of Syk kinase in tumor cells does not appear to reverse the transformed phenotype of tumor cells. In contrast, it is suggested in the present invention that Syk acts in the carcinogenic capacity to promote and / or maintain cell proliferation. In this regard regarding the role of Syk, the present disclosure provides a method of treating a cell proliferative disorder comprising administering to a subject an amount of a Syk kinase inhibitor compound effective to treat the cell proliferative disorder. provide.

  In addition, it is also noted herein that some Syk inhibitor compounds, such as members of the 2,4-pyrimidinediamine compounds described in more detail below, may also act as inhibitors of Flt-3 kinase. It is shown. As a result, in some embodiments, a Syk inhibitor compound having this dual or multi-action inhibition profile (ie, Syk / Flt-3 kinase inhibitory activity) that inhibits Flt-3 kinase as well as Syk kinase is an aberrant Flt. Find uses for the treatment of cell proliferative disorders associated with -3 receptor kinase activity. The combination of both Syk kinase and Flt-3 kinase and the development of hematopoietic cells supports the use of such Syk / Flt-3 kinase inhibitor compounds for the treatment of hematopoietic neoplasms.

  In the description of the method of the present invention, the terms used have their ordinary and common meanings unless otherwise specified herein.

“Syk” or “Syk kinase” refers to a 72 kDa non-receptor (cytoplasmic) spleen protein tyrosine kinase expressed in B-cells and other hematopoietic cells. Syk kinase is characterized by two consensus Src-Homolog 2 (SH2) domains in tandem that bind to a phosphorylated immunoreceptor tyrosine-based activation motif (“ITAM”), a “linker” domain, and a catalytic domain. (For an overview, see Sada et al., 2001, J. Biochem. (Tokyo) 130: 177-186, and also Turner et al., 2000, Immunology Today 21: 148-154, and Wong et al., 2004 Expert Opin Investig Drugs 13 (7): See 743-62). Syk kinase is also important for tyrosine phosphorylation of multiple proteins that regulate key pathways leading from immune receptors, such as Ca ²⁺ mobilization, and the mitogen-activated protein kinase (MAPK) cascade, and degranulation . Syk kinase also plays an important role in integrin signaling in neutrophils (see, eg, Mocsai et al., 2002, Immunity 16: 547-558). Syk kinases include kinases from any animal species recognized as belonging to the Syk family. This animal species includes, but is not limited to, homosapiens, monkeys, cows, pigs, rodents and the like. Specifically, both naturally occurring and artificial isoforms, splice variants, allelic variants, and mutants are included. The amino acid sequence of such Syk kinase can be obtained from GENBANK. A specific example of mRNA encoding different isoforms of human Syk kinase is GENBANK accession number gil21361552 | ref | NM 003177.2, gil 492899 | emb | Z29630.1 | HSSYKPTK [494899], and gil 503258 | gb | BC011399.1 | BC011399 [15030258], which are incorporated herein by reference.

“Flt-3” or “Flt-3 receptor tyrosine kinase” refers to a receptor tyrosine kinase that binds a flt-3 ligand (Flt-3L or FL). Flt-3 is also known as Fms-like tyrosine kinase 3, FLK-2 (fetal liver kinase-2) and STK-1 (human stem cell kinase-11) (eg Mathews et al., 1991, Cell. 65: 1143-1152; Rosnet et al., 1991, Oncogene.6: 1641-1650). Flt-3 has sequence similarity to members of the class III receptor tyrosine kinase (RTKIII) family, a subset of which includes, among others, FMS, platelet derived growth factor receptor (PDGFR), and Kit (Rosnet et al., 1993, Crit Rev Oncog. 4: 595-613). Flt-3 polypeptides in mice and humans are 1000- and 993-amino acid proteins, respectively, and are known to be expressed in immature hematopoietic cells, placenta, gonads, and brain. As mentioned above, Flt-3 has a postulated role in the development of pluripotent stem cells and B cells. Like other members of class III receptor tyrosine kinases, Flt-3 is separated into two cells separated by an extracellular domain, a transmembrane (TM) domain, a proximal membrane (JM) domain, and a kinase insertion (KI) domain. Characterized by internal tyrosine kinase (TK1 and TK2) domains, and five immunoglobulin-like repeats in the C-terminal domain (Agnes et al., 1994, Gene 145: 283-288; 2004, Griffith et al., Molecular Cell 13: 169-178 ). Flt-3 kinase includes kinases from any animal species recognized as belonging to the Flt-3 receptor family, including but not limited to homosapiens, monkeys, cows, pigs, rodents, and the like. Specifically, both naturally occurring and artificial isoforms, splice variants, allelic variants, and mutants are included. The amino acid sequence of such Syk kinase can be obtained from GENBANK. Specific examples of nucleic acid sequences and corresponding amino acid sequences of different Flt-3 kinases are, inter alia, human (accession number NM 004119.1), chimpanzee (accession number 452508; XM 509601.1; XP 509601.1), dog (NM 001020811.1; NP 0010186647.1), and mouse (accession number 142255; NM 010229.11; NP AK0458654; AK1492924; AK1636404; BC1090034; BC1090044; L361634; M646894; X593984), which are incorporated herein by reference.

  Some embodiments of Flt-3 variants are associated with certain hematopoietic cell proliferative disorders. “Flt-3 ITD” refers to a variant of Flt-3 that has an internal tandem duplication (ITD) in the proximal membrane (JM) domain (Nakao et al., 1996, Leukemia 10: 1911-1918; Griffith et al., the above). The number of duplicated JM domains in Flt-3 ITD varies from subject to subject, but is in-frame and generally results in proteins with abnormal (ie, increased) tyrosine kinase activity. Flt-3 ITD type mutants are often associated with acute myeloid leukemia and myelodysplastic syndromes. Another type of variant is the “Flt-3 activation loop mutation”, which has an amino acid sequence change (when compared to the wild type) in the activation loop of the second tyrosine kinase (TK2) domain. Refers to mutants. Without being bound by theory, this activation loop appears to function in preventing access to the kinase domain of adenosine triphosphate (ATP) and substrates, thereby providing an inhibitory effect on the kinase . As a result, activation loop mutations can result in Flt-3 kinase forms with constitutive activity. An exemplary activation loop variant is D835A, which is observed in acute myeloid leukemia, myelodysplastic syndrome, and acute lymphoblastic leukemia (Yamamoto et al., 2001, Blood 97: 2434-2439; Griffin, JD, 2001, Blood 97: 2193a). Other exemplary activation loop variants associated with cell proliferative disorders in humans included, among others, D835Y, D835A, D835E, D835H, D835N, D835V, D835del, and I836del.

  “Cell proliferative disorder” refers to a disorder characterized by abnormal growth of cells. A proliferative disorder does not include restrictions on cell growth rate, but merely shows a loss of normal control that affects growth and cell division. Thus, in some embodiments, proliferative disorder cells may have the same cell division rate as normal cells, but do not respond to signals that limit such growth. Within the area of a “cell proliferative disorder” is a neoplasm or tumor, which is an abnormal growth of tissue. Cancer refers to any of a variety of malignant neoplasms characterized by the growth of cells that have the ability to invade surrounding tissues and / or metastasize to new colonization sites.

  A “hematopoietic neoplasm” refers to a cell proliferative disorder caused by cells of the hematopoietic lineage. In general, hematopoiesis is a physiological process in which undifferentiated cells or stem cells develop as various cells found in peripheral blood. In the early stages of development, hematopoietic stem cells, typically found in the bone marrow, undergo a series of cell divisions to form pluripotent progenitors that are responsible for the two major developmental pathways, the lymphoid and myeloid lineages To do. Myeloid lineage progenitor cells differentiate into three major sub-branches, consisting of red blood cells, megakaryocytes, and granulocyte / monocyte development pathways. An additional pathway results in the formation of dendritic cells involved in antigen presentation. The red blood cell lineage gives rise to red blood cells, while the megakaryocyte lineage gives rise to platelets. The committed cells of the granulocyte / monocyte lineage divide into granulocyte or monocyte development pathways, the former pathway resulting in the formation of neutrophils, eosinophils, and basophils, the latter pathway being blood Produces medium monocytes and macrophages.

  The committed progenitor cells of the lymphoid lineage develop into a B cell pathway, a T cell pathway, or a non-T / B cell pathway. Similar to the bone marrow lineage, additional lymphatic pathways appear to give rise to dendritic cells that are involved in antigen presentation. B cell progenitor cells develop into precursor B cells (pre-B) that differentiate into B cells responsible for immunoglobulin production. Progenitor cells of the T cell lineage differentiate into precursor T cells (pre-T), which develop based on the influence of certain cytokines and are cytotoxic or helper / inhibitor T involved in cell-mediated immunity. Become a cell. The non-T / B cell pathway results in the generation of natural killer (NK) cells. A hematopoietic cell neoplasm may include cells of any stage of hematopoiesis, including hematopoietic stem cells, pluripotent progenitor cells, oligopotent committed progenitor cells, progenitor cells, and mature differentiated cells To do. The category of hematopoietic neoplasia may generally follow the description and diagnostic criteria used by those skilled in the art (see, eg, International Classification of Disease and Related Health Problems (ICD 10), World Health Organization (2003)). Hematopoietic neoplasms also have molecular features such as cell surface markers and gene expression profiles, cell phenotypes indicated by abnormal cells, and / or chromosomal abnormalities characteristic of certain hematopoietic neoplasms (eg deletions, translocations, insertions, etc.) ), For example, based on the Philadelphia chromosome found in chronic myelogenous leukemia. Other classifications include National Cancer Institute Working Formulation (Cancer, 1982, 49: 2112-2135) and Revised European-American Lymphoma Classification (REAL).

  “Lymphoid neoplasm” refers to a proliferative disorder involving cells of the hematopoietic lymphoid lineage. Lymphoid neoplasms can result from hematopoietic stem cells, as well as lymphoid committed progenitor cells, progenitor cells, and terminally differentiated cells. These neoplasms can be subdivided based on the phenotypic attributes of abnormal cells, or the differentiation state caused by abnormal cells. Subdivision includes, among others, B cell neoplasms, T cell neoplasms, NK cell neoplasms, and Hodgkin lymphoma.

  “Bone marrow neoplasm” refers to a proliferative disorder of cells of the hematopoietic bone marrow lineage. Neoplasms can result from hematopoietic stem cells, bone marrow committed progenitor cells, progenitor cells, and terminally differentiated cells. Myeloid neoplasms can be subdivided based on the phenotypic attributes of abnormal cells or the differentiation state caused by abnormal cells. Subdivision includes, among others, myeloproliferative diseases, myelodysplastic / myeloproliferative diseases, myelodysplastic syndromes, acute myelogenous leukemia, and acute biphenotypic leukemia.

  “Virus-mediated tumor” refers to a neoplasm or tumor associated with viral infection, or the activity of a virus-encoded product. Neoplasms can be due to the presence of latent viruses integrated into the cell genome or due to the activity of virus-related gene products. Viral infection does not have to be closely correlated with tumor formation in time in that the incubation period can extend to months or years before the development of the tumor phenotype. Since the treatment in the present invention is directed to the use of Syk inhibitors, applicable virus-related tumors are those in which viral regulation of Syk activity correlates with abnormal cell proliferation. Any virus, including RNA and DNA viruses whose epithelial activation is the result of viral infection and episomally present or integrated into the cell genome, is targeted using the methods of the invention. be able to.

  “Tumor metastasis” refers to the ability of tumor cells to migrate from the original tumor site and colonize in other tissues. Tumors formed from spread cells are called “secondary tumors” and contain cells similar to those in the original “primary” tumor. Metastatic tumors are typically formed by the migration of tumor cells from the original tumor site through the blood and lymph system to other tissues.

“Syk-mediated integrin signaling” refers to signal transduction of cell surface integrins that occurs through interaction with Syk kinase. Integrins comprise an expanded family of cell surface adhesion receptors that bind extracellular matrix and cell surface ligands. Structurally, integrins are heterodimeric proteins consisting of α and β chains, each subunit having an extracellular domain, a single transmembrane domain, and a cytoplasmic domain. The α subunit is generally composed of approximately 7 tandem repeats, with a subset of repeats containing a putative metal binding sequence of the general structure DxDxDGxxD, where x is any amino acid. Two groups of integrins can be characterized by the α subunit. That is, one containing the “A” domain and one having a proteolytic cleavage site. The β subunit contains a conserved region of about 200 amino acids in the extracellular domain, which is a region having structural similarity to the “A” domain of the α subunit and similar to that found in laminin. Characterized by another region with growth factor (EGF) -like repeats (eg, Xiong et al., 2003, Blood, 102 (4): 1155-1159). Integrin activity can regulate intracellular Syk, or conversely, integrin function can be regulated through the activity of Syk. In some instances, it is generally understood that integrins require intracellular activation to bind their cognate ligand (inside-out activation). Integrins that modulate or are regulated by Syk are, inter alia, β1-integrins (Lin et al., J. Biol. Chem. 1995, 270 (27): 16189-97), such as α ₂ b ₁ (Keelly et al. , 1996, J. Biol. Chem. 271 (43): 26668-76), β2-integrin, and β3-integrin (Woodside et al., 2001, Curr Biol. 11 (22): 1799-804), eg, α _IIb β3. (Clark et al., J. Biol. Chem. 1994, 269 (46): 28859-64). For example, Syk is thought to bind directly to the integrin β3 cytoplasmic tail through the SH2 domain. However, unlike Syk binding to ITAM, the interaction with β3 integrin appears independent of the phosphotyrosine binding function of the tandem SH2 domain.

  In general, cell proliferative disorders treatable with the compounds disclosed herein relate to any disorder characterized by abnormal cell proliferation. These include various tumors and cancers that are benign or malignant, metastatic or non-metastatic. Specific characteristics of cancer, such as tissue invasiveness or metastasis, can be targeted using the methods described herein. Cell proliferative disorders include a variety of cancers, which include, among others, breast cancer, ovarian cancer, renal cancer, gastrointestinal cancer, kidney cancer, bladder cancer, pancreatic cancer, lung squamous carcinoma (lung squamous carcinoma). ), And adenocarcinoma.

  In some embodiments, the treated cell proliferative disorder is a hematopoietic neoplasm, which is an abnormal growth of hematopoietic cells. Hematopoietic malignancies can have their origin in pluripotent stem cells, pluripotent progenitor cells, oligopotent committed progenitor cells, precursor cells, and terminally differentiated cells involved in hematopoiesis. Some hematological malignancies are thought to result from hematopoietic stem cells that have the ability to self-renew. For example, cells that can develop specific subtypes of acute myeloid leukemia (AML) upon transplantation show cell surface markers of hematopoietic stem cells, suggesting that hematopoietic stem cells are involved as a source of leukemia cells . Blast cells that do not have cell markers characteristic of hematopoietic stem cells appear to be unable to establish tumors upon transplantation (Blaire et al., 1997, Blood 89: 3104-3112). The stem cell origin of certain hematological malignancies is also supported by the observation that certain chromosomal abnormalities associated with certain types of leukemia are found in normal cells of the hematopoietic lineage as well as leukemic blasts. For example, the reciprocal translocation t (9q34: 22q11) associated with about 95% of chronic myelogenous leukemia appears to be present in cells of the bone marrow, erythrocytes, and lymphoid lineages, indicating that this chromosomal abnormality is hematopoietic. It is derived from stem cells. A subgroup of cells in some types of CML exhibits a cell marker phenotype of hematopoietic stem cells.

  Hematopoietic neoplasms are often derived from stem cells, but committed progenitor cells, or more terminally differentiated cells of the developmental lineage, can also be a source of several leukemias. For example, forced expression of the fusion protein Bcr / Abl (associated with chronic myeloid leukemia) in common myeloid progenitor or granulocyte / macrophage progenitor cells produces a leukemia-like condition. In addition, some chromosomal abnormalities associated with leukemia subtypes are not seen in cell populations with the hematopoietic stem cell marker phenotype, but are found in cell populations that show markers of a more differentiated state of the hematopoietic pathway ( Turhan et al., 1995, Blood 85: 2154-2161). In this way, committed precursor cells and other differentiated cells have limited potential for cell division, while leukemia cells may have acquired the ability to grow unregulated, In such cases, it mimics the self-renewal characteristics of hematopoietic stem cells (Passegue et al., Proc, Natl. Acad. Sci. USA, 2003, 100: 11842-9).

  In some embodiments, the treated hematopoietic neoplasm is a lymphoid neoplasm, and the abnormal cells are derived from and / or exhibit the characteristic phenotype of cells of the lymphoid lineage. Lymphoid neoplasms can be subdivided into B-cell neoplasms, T and NK-cell neoplasms, and Hodgkin lymphoma. B-cell neoplasms can be further subdivided into precursor B-cell neoplasms and mature / peripheral B-cell neoplasms. An exemplary B-cell neoplasm is a precursor B-lymphoblastic leukemia / lymphoma (precursor B-cell acute lymphoblastic leukemia), while an exemplary mature / peripheral B-cell neoplasm is B -Cell chronic lymphocytic leukemia / small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoblastic lymphoma, splenic marginal band B-cell lymphoma, hairy cell leukemia, plasma cell myeloma / plasma cell MALT type extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal large B-cell lymphoma, Primary exudative lymphoma, and Burkitt lymphoma / Burkitt cell leukemia. T-cell and NK-cell neoplasms are further subdivided into precursor T-cell neoplasms and mature (peripheral) T-cell neoplasms. An exemplary precursor T-cell neoplasm is a precursor T-lymphoblastic lymphoma / leukemia (precursor T-cell acute lymphoblastic leukemia), while an exemplary mature (peripheral) T-cell neoplasm The organism is T-cell prolymphocytic leukemia, T-cell granulocyte leukemia, aggressive NK-cell leukemia, adult T-cell lymphoma / leukemia (HTLV-1), extranodal NK / T-cell lymphoma Nasal cavity type, intestinal disease type T-cell lymphoma, hepatosplenic γ-δ T-cell lymphoma, subcutaneous lipohistitis-like T-cell lymphoma, fungal mycosis / Sezary syndrome, undifferentiated large cell lymphoma, T / null cell Primary skin type, peripheral T-cell lymphoma, uncharacterized, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, T / null cell, primary systemic type. The third member of the lymphoid neoplasm is Hodgkin's lymphoma, also called Hodgkin's disease. This type of exemplary diagnosis that can be treated with these compounds includes, among other things, nodular lymphocyte-dominated Hodgkin lymphoma, and various conventional types of Hodgkin's disease, these exemplary members being nodular Sclerosis Hodgkin lymphoma (grades 1 and 2), lymphocyte-rich conventional Hodgkin lymphoma, mixed cytoplasmic Hodgkin lymphoma, and lymphocyte-depleted Hodgkin lymphoma. In various embodiments, any of the lymphoid neoplasms associated with abnormal Syk activity can be treated with a Syk inhibitor compound.

  In some embodiments, the hematopoietic neoplasm to be treated is a bone marrow neoplasm. This group comprises a large class of cell proliferative disorders that are involved or exhibit the characteristic phenotype of cells of the myeloid lineage. Myeloid neoplasms can be subdivided into myeloproliferative diseases, myelodysplastic / myeloproliferative diseases, myelodysplastic syndromes, and acute myeloid leukemia. Exemplary myeloproliferative diseases are chronic myelogenous leukemias (eg Philadelphia chromosome positive (t (9; 22) (qq34; q11)), chronic neutrophilic leukemia, chronic eosinophilic leukemia / hypereosinophils Sexual syndrome, chronic idiopathic myelofibrosis, polycythemia vera, and essential thrombocythemia Exemplary myelodysplastic / myeloproliferative diseases are chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, An exemplary myelodysplastic syndrome is refractory anemia with or without cyclic iron blasts, refractory cytopenia with multiple lineage dysplasia (bone marrow). Dysplastic syndrome), refractory anemia with excessive buds (myelodysplastic syndrome), 5q-syndrome, and myelodysplastic syndrome with t (9; 12) (q22; p12) (TEL-Syk fusion; Example In Kuno et al, 2001, Blood 97:. 1050 see) In various embodiments, none of the associated with aberrant Syk activity myeloid neoplasms can be treated with the Syk inhibitory compounds.

  In some embodiments, Syk inhibitor compounds can be used to treat acute myeloid leukemia (AML). This represents a large class of bone marrow neoplasia with its own subdivision of the disorder. These sub-categories include, among other things, AML with recurrent cytogenetic translocations, AML with multilineage dysplasia, and other AML that do not fall into other categories. Exemplary AMLs with recurrent cytogenetic translocations include AML with t (8; 21) (q22; q22), AML1 (CBF-α) / ETO, acute promyelocytic leukemia (t (15; 17), among others. ) AML with (q22; q11-12) and mutant PML / RAR-α), abnormal bone marrow eosinophils (inv (16) (p13q22) or t (16; 16) (p13: q11), CBFb AML with / MYH11X) and AML with 11q23 (MLL) abnormalities. Exemplary AML with multilineage dysplasia is one that is or is not associated with previous myelodysplastic syndromes. Other acute myeloid leukemias not classified in any definable group are minimally differentiated AML, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monocytic leukemia , Acute erythroleukemia, acute megakaryocytic leukemia, acute basophilic leukemia, and acute panmyelosis with myelofibrosis.

  In other embodiments, cell proliferative disorders that can be targeted with Syk kinase inhibitors include virus-mediated tumors. These can result from infection of the cells with oncogenic viruses that have the ability to transform normal cells into tumor cells. Since viral infection rates far exceed the actual number of cell transformations, virus-mediated transformation generally works with other cellular factors to generate transformed tumor cells. In this way, virus-mediated tumors do not require that the virus be the sole causative agent of a cell proliferative disorder, but rather the viral infection or the permanent presence of the virus is associated with tumor development. You need to be. In general, tumors whose causative agent is a virus typically have a continuous expression of a limited number of viral genes, and these viral oncogenes are either part of the viral infection or are persistent Through and disrupts normal cellular gene expression and signaling pathways. Without being bound by theory, the viral oncogenes involved in cell transformation appear to disrupt four major cellular processes: cell surface receptors that interact with growth factors and extracellular matrix, Nuclear proteins, including transmembrane signaling networks, cytosolic elements such as soluble proteins and second messengers, and DNA binding proteins and factors that function directly and indirectly in gene regulation and replication. Because certain virus-encoded proteins target specific cellular components as part of the transformation process, application of Syk inhibitors may be appropriate when the viral component targets the activity of Syk kinase.

In some embodiments, the virus-mediated tumor treatable with the compounds disclosed herein is any virus that encodes an immunoreceptor tyrosine-based activation motif (ITAM) that can modulate Syk activity. Are related. This motif refers to a conserved amino acid sequence motif that functions by interacting with and activating the non-receptor tyrosine kinase, as described above. ITAM motifs are found, inter alia, in the β and γ chains of FcεRI, the ε subunit of the T cell receptor, and immunoglobulin β (Igβ), and Igα of the B cell receptor. The canonical sequence motif is typically Yxx (L / I) x _6-8 Yxx (L / I), where x represents any amino acid. In general, the tyrosine residue in this motif is involved in ITAM signaling and is a substrate for phosphorylation by the Src family of kinases. The phosphorylated form of ITAM functions as an interaction site for signal transduction proteins such as SH2 (src homolog domain) containing Syk / ZAP-70 kinase. In addition to its presence in the cell surface molecules of diverse cells, ITAM sequences have been identified in virally encoded proteins. In view of the description herein pointing out the function of Syk kinase as an oncogene, tumors associated with viruses carrying a gene encoding a protein having an ITAM sequence can be treated with a Syk inhibitor compound. .

  Accordingly, in some embodiments, virus-mediated tumors treatable with these inhibitory compounds are at a higher incidence among Kaposi's sarcoma (KS) -related herpesviruses, lymphotrophic virus associated with Kaposi's sarcoma, HIV-infected populations. Associated with rare malignant tumors that are discovered. The KS-related herpesvirus encodes a transmembrane protein called KI that has an immunoreceptor tyrosine-based activation motif (ITAM) -like sequence. The KI gene product is thought to act constitutively through its cysteine-rich ectodomain to activate Syk and its related kinase Zap-70 (Lagunoff, M. et al., 1999, Proc. Natl. Acad. Sci. USA 96 (10): 5704-5709). In further support of the method of the present invention, transgenic mice carrying the KI gene appear to increase the incidence of certain sarcomas and lymphoid species in infected animals, indicating a role for KI activity in tumorigenesis. (Prakash et al., 2002, J. Natl. Cancer Inst. 94: 926-35).

In some embodiments, the virus-mediated tumor is associated with Epstein-Barr virus (EBV). Epstein-Barr virus is a member of the Herpesviridae family that replicates in epipharyngeal epithelial cells after primary infection and infects recirculating B lymphocytes. Infection can result in acute infectious monocytosis, also known as glandular fever. Infectious monocytosis is a benign lymphoproliferative disease characterized by transient immunosuppression and abnormal lymphocyte expansion, mostly CD8 ⁺ T cells. In these T cells, EBV establishes a latent but permanent infection, during which time a select set of viral genes are expressed. The entire genome can persist in lymphocytes growing as episomal DNA. EBV infection is associated with Burkitt lymphoma, Hodgkin lymphoma, and adult T-cell leukemia.

  The LMP2A protein encoded by the EBV genome is a transmembrane protein that is thought to play a role in maintaining the latency of the EBV virus after infection. It consists of an extended amino terminal tail, a 12 membrane span domain, and a cytoplasmic domain. The amino terminal region contains an ITAM motif, which allows the interaction of LMP2A with Syk kinase (Fruehling et al., 1997, Virology, 235: 241-251). LMP2A appears to regulate Syk kinase in lymphocytes to promote B-cell survival and maintain latency. Syk plays an important role in signaling pathways that regulate other signaling pathways such as PI-3K, BLNK, and phospholipase γ2, and is involved in enhancing lymphocyte survival, so LMP2A protein, or other Inappropriate Syk activation through virus-mediated effectors may play a role in the induction of abnormal lymphoproliferation (Caldwell et al., 2000, J Virol 74 (19): 9115; Caldwell et al., 1998, Immunity 9 : 405). Thus, inhibition of Syk activity can have a therapeutic effect on cell proliferative disorders associated with EBV virus infection.

In some embodiments, the virus-mediated tumor to be treated with a Syk inhibitor is a human T-cell lymphocytic virus (HTLV-1 virus), a retrovirus in the same type of virus as AIDS virus HIV-1. Associated with viruses. CD8 ⁺ T-cells can also function as a viral reservoir, but the virus is also affinity for CD4 ⁺ T-cells. HTLV-1 infection is associated with, among other things, adult T-cell leukemia / lymphoma (ATLL), and several other lymphocyte disorders. During HTLV-1 infection, Syk is expressed in infected cells, while there is no expression of the Syk-related kinase ZAP-70 (Weil et al., 1999, J. Virol. 73 (5): 3709-17). Several dysregulated kinases, including Syk, have been implicated in HTLV-1-mediated induction of adult T-cell leukemia.

  In some embodiments, the virus-mediated tumor is associated with breast cancer virus (MTV). The ITAM sequence is found in the Env gene of murine mammary tumor virus (MMTV), a B-type retrovirus identified as a pathogenic agent for mouse breast cancer. Mouse mammary epithelial cells transfected with the MMTV Env gene display properties of the transformed phenotype, such as colonization in soft agar, and invasion into basement membrane preparations (Katz et al., 2005, J Exp Med 201 (3): 431-9). Murine mammary tumor virus-like sequences are also present in human cancers such as breast cancer and T-cell lymphoma (Wang et al., 2000, Clinical Cancer Res. 6: 1273-1278) for reasons related to tumor formation. This is because these sequences are not observed in the majority of normal breast tissue. In this way, tumors associated with MTV can be treated with Syk kinase inhibitors.

  It should be understood that the use of Syk inhibitor compounds for the treatment of virus mediated tumors is not limited to tumors associated with the previously identified viruses. As described, any tumor associated with an oncogenic virus in which Syk is activated as part of its oncogenic mechanism can be obtained using a Syk inhibitor compound, whether this is with or without an ITAM sequence. Can be targeted.

  In some embodiments, cell proliferative disorders that can be targeted with these inhibitors include cell proliferative disorders associated with aberrant Flt-3 kinase activity. The term “abnormal Flt-3 kinase activity” refers to an activity that is not normal in view of: That is, what is the normal level of Flt-3 kinase activity for wild-type Flt-3 kinase or in normal Flt-3 expressing cells, tissues, organs, or organisms? From this point of view. Abnormal Flt-3 kinase activity is due to protein mislocalization (spatial expression), increased or decreased enzyme activity (directly or indirectly), or transient expression changes (ie developmental expression). sell.

  In some embodiments, the aberrant kinase activity is associated with a variant of Flt-3. In some embodiments, these variants are characterized by increased Flt-3 receptor kinase activity. As used herein, “increased kinase activity” refers to that observed for wild-type Flt-3 kinase or in normal Flt-3 expressing cells, tissues, organs, or organisms. Also refers to high kinase activity. Exemplary increased kinase activity is found in cells with certain Flt-3 variants, such as Flt-3 ITD, and Flt-3 activation loop mutations.

  Since aberrant Flt-3 kinase activity is observed in many different types of hematopoietic neoplasms, in some embodiments, a Syk / Flt-3 inhibitory compound is responsible for the presence of aberrant Flt-3 kinase. It can be used to treat characteristic hematopoietic neoplasms. Thus, in some embodiments, hematopoietic neoplasms treatable with a Syk / Flt-3 inhibitor compound include, among others, acute myeloid leukemia, B-progenitor cell acute lymphoblastic leukemia, T-cell Acute lymphoblastic leukemia and chronic myelogenous leukemia (CML) that has been diagnosed or suspected of the presence of abnormal Flt-3 in the subject may be included. However, one of ordinary skill in the art should understand that these treatments can be applied without such information, and that these inhibitory compounds can be given prophylactically to increase the probability of survival. is there. In addition, one of ordinary skill in the art can apply the Syk / Flt-3 inhibitory compounds herein to other cell proliferative disorders in which abnormal Flt-3 kinase activity has been detected or suspected.

  In other aspects, the present disclosure is directed to the treatment of tumor metastases through the use of Syk kinase inhibitors. Metastasis is a feature of malignant tumor cells in which tumor cells detach from their site of origin and then spread and colonize at other sites. These secondary tumors can form in tissues that are not associated with the cells from which the tumor cells are derived. It is the formation of these secondary tumors by metastasis that appears to be the leading cause of mortality from malignant forms of cancer. Metastasis begins when malignant cells leave the primary tumor, enter the blood or lymph system, and then migrate to other colonization sites. In general, normal cells do not detach and invade other tissues by various signals that inhibit foreign cells from adhering to each other, as well as signals between cells that inhibit cell growth. However, cell transformation alters these normal regulatory programs so that tumor cells interact with local tissue cells to modify the local extracellular matrix, stimulate migration, and promote proliferation and survival. To do. Modifications of cell adhesion molecules (CAM) such as immunoglobulins and calcium-dependent cadherin family and integrin members appear to play an important role in invasion and metastasis. For example, modification of N-CAM from a highly adherent isoform to a less adherent form, along with its down-regulation, can result in invasive pancreatic cancer.

  Another class of adhesion proteins involved in tissue invasion and metastasis are integrins. As described above, integrins form a diverse class of cell surface molecules. Permutations within more than 22 integrin subtypes can also create a flexible system for altering cell interactions in response to different cellular signaling states or changing external environments. Integrins interact dynamically with extracellular ligands depending on the activation state of the integrin. This is controlled by intracellular signals that modify integrin affinity and binding activity (ie, inside-out signaling). Conversely, interactions between integrins and extracellular ligands can trigger signaling cascades that affect cell adhesion properties and cellular responses to the changing extracellular environment (ie, outside-in signaling). Changes in integrin subunit expression induce or inhibit invasive and metastatic growth, suggesting that these proteins are involved as important determinants of these processes (Guo et al., 2004, Nat. Rev. Mol.Cell Biol.5 (10): 816-26; Jin et al., 2004, Br.J. Cancer, 90: 561-565). For example, the integrin αvβ3 is required for angiogenesis and its upregulation correlates with tumor invasiveness and metastatic potential (Liapis et al., 1996, Diag. Mol. Pathol. 23: 127-135). Cultured metastatic breast cancer cells show constitutive expression of αvβ3 (Pechur et al., 2002, FASEB J.16: 1266-1268), while, for example, RGD peptides that compete for binding of integrin to its natural substrate Inhibition of αv subunit containing integrin activity through the use of analogs can reduce tumor metastatic potential without affecting cell growth properties (Krinsten et al., 2004, Clin. Exp. Metastasis 21 ( 2): 129-38; Harms et al., 2004, Clin. Exp. Metastasis 21 (2): 119-28). Similarly, overexpression of β1 integrin may destroy adhesive junctions that serve to keep cells attached to the basement membrane.

Syk kinase activity is expressed on cells of the hematopoietic lineage, but is associated with various integrins that are also expressed in non-hematopoietic cells as well. Syk kinase is found in lung epithelial cells (Ulanova et al., 2004, Am. J. Physiol. Lung Cell Mol. Physiol. 288: L497-L507) and monocytes (Lin et al., 1995, J. Biol. Chem. 270 (27). 16189-97), β2 integrin signaling in granulocytes / neutrophils (Miura et al., 2000, Blood 96 (5): 1733-9; Kusumoto et al., 2001, Microbiol. Immunol. 45 ( 3): 241-8), and β3 integrin signaling in platelet activation and cell adhesion (Gao et al., 1997, EMBO J. 16 (21): 6414-25). Given the relationship shown in the present invention between Syk kinase activity and tumorigenesis, the use of Syk kinase inhibitors in reducing the invasive and metastatic properties of tumors is associated with Syk kinase activity. (Mocsai et al., 2002, Immunity 16 (4): 547-58). Thus, in some embodiments, inhibitors of Syk kinase can be used to modulate tumor metastatic properties mediated by integrin activity. In some embodiments, Syk kinase inhibitors can be used to reduce tumor cell tissue invasiveness and metastatic potential affected by β1 integrin (Lin et al., 1995, J. Biol. Chem). 270: 16189-16197; Kusumoto et al., Microbiol Immunol. 2001, 45 (3): 241-8; Ortiz-Stern et al., 2005, J. Leukoc Biol. (Epub)). An exemplary integrin of this type is integrin α ₂ b ₁ .

In some embodiments, these Syk kinase inhibitors can be used to reduce tumor cell tissue invasiveness and metastatic potential affected by the activity of β2 integrin (CD18) (Willeke et al., 2003). , J Leukoc.Biol.74 (2): 260-9). These include CD11a / CD18, CD11b / CD18, CD11c / CD18, and CD11d / CD18, among others. In further embodiments, these Syk kinase inhibitors can be used to reduce tumor cell tissue invasiveness and metastatic potential affected by the activity of β3 integrin. Exemplary integrins of this type are α _IIb β ₃ and α _v β ₃ .

  Various tumor types capable of metastasis can be treated with these Syk inhibitor compounds. Such tumors include, by way of example and not limitation, breast cancer, ovarian cancer, renal cancer, gastrointestinal cancer, kidney cancer, bladder cancer, pancreatic cancer, lung squamous cancer, and adenocarcinoma (eg, Felding-Habermann). Et al., 2001, Proc Natl Acad Sci USA 98 (4): 1853-8). Therapeutic treatment to reduce metastasis of established tumors can follow the diagnosis of metastases. If no diagnosis of metastasis has been made, the inhibitory compound can be administered prophylactically to reduce the probability of metastasis.

  These Syk inhibitor compounds can be used independently of any other treatment or can be used in combination with other cancer therapies, including surgery, radiation use, or other chemotherapy Should be understood. Thus, in some embodiments, these Syk kinase inhibitors can be used in combination with other chemotherapeutic agents. Combination therapy with Syk inhibitors may target different cellular components by appropriate selection of a second chemotherapeutic agent. For example, a Syk inhibitor can be used in some embodiments to limit the metastatic potential of tumor cells, while another chemotherapeutic agent is used to remove or kill abnormal cells. Can be used.

  A variety of chemotherapeutic agents can be used in combination with Syk kinase inhibitors to treat cell proliferative disorders. These chemotherapeutic agents may be common cytotoxic agents or may target specific cellular molecules. Various classes of cancer chemotherapeutic agents include, among others, antimetabolites, agents that react with DNA (eg, alkylating agents, coordination compounds, etc.), inhibitors of transcriptases, topoisomerase inhibitors, DNA minor groove binding compounds, Includes anti-mitotic drugs (eg vinca alkyloids), antitumor antibiotics, hormones, and enzymes. Exemplary alkylating agents include, by way of example and not limitation, mechlorotamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, ethyleneimine, methylmelamine, alkyl sulfonates (eg, busulfan), and carmustine. Exemplary antimetabolites include, by way of example and not limitation, the folate analog methotrexate; the pyrimidine analog fluorouracil, cytosine arabinoside; and the purine analogs mechatoppurine, thioguanine, and azathioprine. Exemplary vinca alkyloids include, by way of example and not limitation, vinblastine, vincristine, paclitaxel, and corsicin. Exemplary anti-tumor antibiotics include, by way of example and not limitation, actinomycin D, daunorubicin, and bleomycin. An exemplary enzyme effective as an anti-neoplastic agent is L-asparaginase. Exemplary coordination compounds include, but are not limited to, cisplatin and carboplatin. Exemplary hormones and hormone-related compounds include, but are not limited to, adrenocorticosteroid prednisone and dexamethasone; aromatase inhibitors aminoglutethimide, formestane, and anastrozole; progestin compounds hydroxyprogesterone caproate, medroxy Progesterone; and the antiestrogen compound tamoxifen. Exemplary topoisomerase inhibitors include, by way of example and not limitation, amsacrine (m-AMSA); mitoxantrone, topotecan, irinotecan, and camptothecin.

  These anti-cancer compounds and other useful anti-cancer compounds are described in Merck Index, 13th Ed. (O'Neil, MJ, et al., Ed) Merck Publishing Group (2001), and Goodman and Gilman The Pharmaceutical Basis of Therapeutics, 10th Edition, Hardman. G. And Limbird, L .; E. eds. pg. 1381-1287, McGraw Hill, (1996). Both of these documents are hereby incorporated by reference.

  Other anti-proliferative compounds useful in combination with Syk inhibitors include, but are not limited to, antibodies directed against growth factor receptors (eg anti-Her2); cytokines such as interferon-α and interferon- γ, interleukin-2, and GM-CSF; and antibodies for cell surface markers (eg, anti-CTLA-4, anti-CD20 (rituximab); anti-CD33). When antibodies against cell surface markers are used, chemotherapeutic agents can be conjugated to them for specific targeting to tumor cells. Suitable conjugates include radioactive compounds (eg, radioactive metals conjugated to antibody conjugated chelators), cytotoxic compounds, and drug activating enzymes (eg, allinases, peptidases, esterases, catalytic antibodies, etc.) (eg, Arditi). 2005, Mol. Cancer Therap. 4 (2): 325-331; see US Pat. No. 6,258,360, which are incorporated herein by reference).

  In some embodiments, the Syk inhibitor can be used in conjunction with a second kinase inhibitor that targets Syk, or an oncogenic kinase different from Syk and Flt-3. If Syk inhibitors are disclosed herein for the treatment of hematopoietic neoplasms, other compatible kinase inhibitors that have been used for the treatment of hematopoietic neoplasms may also be used. it can. In some embodiments, the second kinase inhibitor is an inhibitor of AbI kinase. Chronic myeloid leukemia is a myeloid neoplasm characterized by malignant growth of leukemic stem cells in the bone marrow. The majority of chronic myeloid leukemia is associated with cytogenetic abnormalities defined by the reciprocal translocation t (9; 22) (q34; q11). This chromosomal abnormality results in the generation of a BCR / ABL fusion protein with activated kinase activity. This inhibitor of fusion protein kinase activity may be effective in the treatment of chronic myelogenous leukemia, although resistant forms may develop during continuous treatment. The use of a Syk kinase inhibitor in combination with an AbI kinase inhibitor may reduce the chance of resistant cells by targeting a different cellular process than that targeted by the second kinase inhibitor. An exemplary Abl kinase inhibitor is 2-phenylaminopyrimidine, also known as imatinib mesylate and Gleeve®. Thus, in some embodiments, these Syk kinase inhibitors can be used in combination with the Abl kinase inhibitor 2-phenylaminopyrimidine and its derivatives. In other embodiments, the second kinase inhibitor may be pyridol [2-3-d] pyrimidine and its derivatives, which were originally identified as inhibitors of Src kinase. In yet other embodiments, the second kinase inhibitor may be tyrphostin and its derivatives (eg, adaphostin), which may affect the association of the kinase with its substrate. Other kinase inhibitor compounds will be apparent to those skilled in the art.

  As further described herein, administration of other chemotherapeutic agents may be in the form of a composition or may be additionally administered in combination with a Syk inhibitor. The chemotherapeutic agent, when given in addition, may be administered simultaneously or sequentially with the administration of the Syk inhibitor.

6.2 Syk Kinase and Syk / Flt-3 Kinase Inhibitors The terms used to describe these compounds in relation to various inhibitors are different definitions given herein or specific Unless otherwise indicated in the references describing the inhibitory compounds, they have their usual and common meaning as used by those skilled in the art.

"Alkyl", alone or as part of another substituent, refers to the stated number of carbons derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene, or alkyne. Saturated or unsaturated, branched, straight chain, or cyclic monovalent hydrocarbon group having atoms (ie C ₁ -C ₆ means 1 to 6 carbon atoms). Typical alkyl groups are methyl; ethyl, such as ethanyl, ethenyl, ethynyl; propyl, such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, Prop-1-en-2-yl, prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl, cycloprop-2-en-1-yl, prop-1-in-1 -Yl, prop-2-yn-1-yl, etc .; butyl, such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutane -1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but 2-en-2-yl, but- , 3-Dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-diene-1 -Yl, but-1-in-1-yl, but-1-in-3-yl, but-3-in-1-yl, and the like. The term “alkyl” specifically includes groups having any degree or level of saturation, ie, groups having only carbon-carbon single bonds, groups having one or more carbon-carbon double bonds, It is intended to include groups having one or more carbon-carbon triple bonds, and groups having a mixture of carbon-carbon single bonds, double bonds, and triple bonds. Where a specific level of saturation is intended, the expressions “alkanyl”, “alkenyl”, and “alkynyl” are used. The expression “lower alkyl” refers to an alkyl group of 1 to 6 carbon atoms.

  “Alkanyl” by itself or as part of another substituent refers to a saturated branched, straight chain, or cyclic alkyl group. Typical alkanyl groups are methanyl; ethanyl; propanyl, such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, and the like; butanyl, such as butan-1-yl, butane-2-yl Including but not limited to yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, and the like. .

  “Alkenyl” by itself or as part of another substituent has at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of the parent alkene. An unsaturated branched, straight chain, or cyclic alkyl group. This group may be in either cis or trans conformation around one or more double bonds. Typical alkenyl groups are ethenyl; propenyl such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-ene 2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2- Methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-diene- 1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl and the like However, it is not limited to these.

  “Alkynyl”, alone or as part of another substituent, refers to an alkyl having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Saturated branched, straight chain, or cyclic alkyl group. Typical alkynyl groups are ethynyl; propynyl such as prop-1-in-1-yl, prop-2-in-1-yl and the like; butynyl such as but-1-in-1-yl, but-1- Examples include, but are not limited to, in-3-yl, but-3-in-yl, and the like.

  “Parent aromatic ring system” refers to an unsaturated cyclic or polycyclic ring system having a conjugated pi electron system. Specifically, within the definition of “parent aromatic ring system”, fused ring systems in which one or more of these rings are aromatic and one or more of these rings are saturated or unsaturated For example, fluorene, indane, indene, phenalene and the like are included. Typical parent aromatic ring systems are: acanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indan Indene, Naphthalene, Octacene, Octaphene, Octaphene, Octalene, Ovalene, Penta-2,4-diene, Pentacene, Pentalene, Pentaphene, Perylene, Phenalene, Phenanthrene, Picene, Preaden, Pyrene, Pyrenelene, Rubicene, Triphenylene, Trinaphthalene, etc. Including, but not limited to.

“Aryl”, alone or as part of another substituent, refers to the number of carbocyclic atoms described by the removal of one hydrogen atom from a single carbon atom of the parent aromatic ring system. (Ie C ₅ -C ₁₄ means a 5-14 carbon ring atom). Typical aryl groups are asanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, Groups derived from naphthalene, octacene, octaphen, octalene, obalene, penta-2,4-diene, pentacene, pentalene, pentaphen, perylene, phenalene, phenanthrene, picene, preaden, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, etc. Including, but not limited to. In a preferred embodiment, the aryl group is (C ₅ -C ₁₄ ) aryl, more preferably (C ₅ -C ₁₀ ). Particularly preferred aryls are cyclopentadienyl, phenyl, and naphthyl.

“Arylalkyl” by itself or as part of another substituent refers to a non-substituted hydrogen atom, typically a terminal or sp ₃ carbon atom, substituted with an aryl group. Refers to a cyclic alkyl group. Typical arylalkyl groups are benzyl, 2-phenylenetan-1-yl, 2-phenyleneten-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2-naphthylethen-1-yl, naphthobenzyl , 2-naphthophenylethane-1-yl, and the like. Where specific alkyl moieties are intended, the terms arylalkanyl, arylalkenyl, and / or arylalkynyl are used. In preferred embodiments, the arylalkyl group is (C ₆ -C ₁₆ ) arylalkyl, for example, the alkanyl, alkenyl, or alkynyl moiety of the arylalkyl group is (C ₁ -C ₆ ) and the aryl moiety is ( a C ₅ -C _10). In particularly preferred embodiments, the arylalkyl group is (C ₆ -C ₁₃ ), for example, the alkanyl, alkenyl, or alkynyl moiety of the arylalkyl group is (C ₁ -C ₃ ) and the aryl moiety is (C ₅ is a -C _10).

  “Parent heteroaromatic ring system” refers to a parent aromatic ring system in which one or more carbon atoms are each independently substituted with the same or different heteroatoms or heteroatom groups. Typical heteroatoms or heteroatomic groups for substituting carbon atoms include, but are not limited to, N, NH, P, O, S, Si, and the like. Specifically, in the definition of “parent heteroaromatic ring system”, a fused ring system in which one or more of these rings are aromatic and one or more of these rings are saturated or unsaturated Is included. For example, arsindole, benzodioxane, benzofuran, chroman, chromene, indole, indoline, xanthene and the like. Similarly, within the definition of “parent heteroaromatic ring system” are included recognized rings, such as benzopyrone, including substituents. Typical parent heteroaromatic ring systems are arsindole, benzodioxane, benzophyrane, benzopyrone, carbazole, β-carboline, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene. , Isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, Pyrrole, pyrrolidine, quinazoline, quinoline, quinolidine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, Riazor, xanthene encompasses the like, but not limited to.

  "Heteroaryl", alone or as part of another substituent, refers to the number of ring atoms described, derived by the removal of one hydrogen atom from a single atom of the parent heteroaromatic ring system. (Ie, “5 to 14 membered” means a monovalent heteroaromatic group which means 5 to 14 ring atoms). Typical heteroaryl groups are acridine, arsindole, carbazole, β-carboline, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline. , Isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, Derived from quinolidine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, etc. Including, but are not limited to. In some embodiments, the heteroaryl group is a 5-14 membered heteroaryl, or a 5-10 membered heteroaryl.

“Heteroarylalkyl”, alone or as part of another substituent, refers to one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp ₃ carbon atom, substituted with a heteroaryl group. An acyclic alkyl group. Where specific alkyl moieties are intended, the terms heteroarylalkanyl, heteroarylalkenyl, and / or heterolylalkynyl are used. In some embodiments, the heteroarylalkyl group is a 6-20 membered heteroarylalkyl, eg, the alkanyl, alkenyl, or alkynyl moiety of the heteroarylalkyl is 1-6 membered, and the heteroaryl moiety is 5- 14-membered heteroaryl. In particularly preferred embodiments, the heteroarylalkyl is a 6-13 membered heteroarylalkyl, for example, the alkanyl, alkenyl, or alkynyl moiety is 1-3 membered and the heteroaryl moiety is 5-10 membered heteroaryl.

“Substituted alkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl” refers to an alkyl, aryl, arylalkyl, heteroaryl, or heteroaryl in which one or more hydrogen atoms are replaced with another substituent Refers to an alkyl group. Exemplary substituents are —OR ′, —SR ′, —NR′R ′, —NO ₂ , —NO, —CN, —CF ₃ , halogen (eg, —F, —Cl, —Br, and —I). , -C (O) R ', - C (O) OR', - C (O) NR ', - S (O) 2 R', - S (O) 2 NR'R '( wherein each R 'Independently includes, but is not limited to, hydrogen and (C ₁ -C ₆ ) alkyl).

  “Prodrug” refers to a derivative of an active compound (drug) that requires transformation in order to release the active agent, eg, in the body. Prodrugs are often pharmacologically inactive until converted to the active drug, but this is not always the case. Prodrugs are typically obtained by masking a functional group in a drug that is thought to be required in part for activity with a pro group (defined below) Which undergoes transformation, eg cleavage, under certain conditions of use, releasing the functional group and thus yielding the active agent. The cleavage of the pro moiety can proceed naturally, for example by a hydrolysis reaction. Or it can be catalyzed or triggered by another agent, such as an enzyme, light, acid, or a change in or exposure to a physical or environmental parameter, such as a change in temperature. This agent may be endogenous to the conditions of use, eg, enzymes present in the cells to which the prodrug is administered, or acidic conditions of the stomach, or it may be supplied externally.

  A wide variety of prodrugs suitable for masking functional groups in active agents to produce prodrugs, as well as the resulting promoieties are well known in the art. For example, a hydroxyl functional group may be masked as a sulfonate, ester, or carbonate promoiety, which can be hydrolyzed in vivo to yield a hydroxyl group. The amino functionality may be masked as an amide, carbamate, imine, urea, phosphenyl, phosphoryl, or sulfenyl pro moiety, which can be hydrolyzed in vivo to yield an amino group. Carboxyl groups may be masked as esters (including silyl esters and thioesters), amides, or hydrazide pro moieties, which can be hydrolyzed in vivo to yield carboxyl groups. Other examples of suitable progroups and their respective promoieties will be apparent to those skilled in the art.

A variety of compounds that inhibit Syk kinase or Syk / Flt-3 kinase activity can be used in the methods described herein. These include inter alia small organic molecules, peptides or proteins, or nucleic acids. As used herein, a “Syk inhibitor” or “Syk kinase inhibitory compound” directly inhibits the activity of Syk kinase itself, or the IC ₅₀ range described herein. Refers to any compound that inhibits the interaction of Syk with other cellular targets required for proper Syk function. Inhibitors as used herein include conventional descriptions of enzyme inhibitors such as competitive, non-competitive, and non-competitive inhibitors. A compound that is a Syk inhibitor is generally about 5 uM or less, about 1 uM or less, about 1 uM or less for Syk kinase activity, eg, the ability of Syk kinase to phosphorylate a synthetic or endogenous substrate, in an in vitro or cellular assay. A compound that exhibits an IC ₅₀ within a range of 500 nm or less, about 100 nm or less, about 50 nm or less, about 10 nm or less, or about 1 nm or less. For example, exemplary Syk inhibitor compounds are disclosed in US patent application Ser. No. 10 / 631,029 and PCT Publication No. WO 2004/014382. One skilled in the art will appreciate that compounds that exhibit a lower IC ₅₀ , eg, an IC ₅₀ within a range that is about 100 nM, 10 nM, 1 nM, or even lower, are useful in the methods of the invention.

In some embodiments, the inhibitory compound may be selective for Syk kinase. The "Syk kinase selective inhibitory compound" refers to compounds which show selectivity for Syk, which is at a defined set of assays, the ratio of IC ₅₀ for relative IC ₅₀ for Syk kinase, see kinase Is defined as In general, a Syk kinase selective inhibitory compound may have a selectivity for Syk kinase that is greater than about 10, greater than about 50, greater than about 100, greater than about 1,000, or more. The reference kinase may be any kinase activity associated with a cell proliferative disorder, such as, but not limited to, Aurora-A, AKT, CDK1 / cyclin B, CDK2 / cyclin A. , CDK3 / cyclin E, CDK5 / p35, CDK6 / cyclin D3, CDK7 / cyclin H / MAT1, CHK1, CHK2, EGFR, c-RAF, RAS, cSRC, Yes, Fyn, Lck, Fes, Lyn, Bmx, FGFR3, GSK3α, GSK3β, P13, IGF-1R, MAPK2, MAPKAP-K2, JNK, MEK1, p70S6K, PAK2, PDGFRα, PDGFRβ, PDK1, PKA, PKCε, PKC, PKD2, VEGF, PRAK, PRAK1, PRAK1 Includes Rsk2, Rsk3, SGK. Various assays for each of these kinases will be apparent to those skilled in the art. For example, Aurora kinase activity can use natural or synthetic substrates (eg, fluorescent peptides, histone H3) in in vitro assays or measurement of phosphorylated products in cells (Walter et al., 2000, Oncogene 19 (42): 4906. 16). Kinase activity can be detected using a variety of research methods. This includes, but is not limited to, immunoprecipitation (eg, Cyclex Aurora A Kinase Assay; MBL Corp, Woburn, MA, USA), mobility shift (eg, Caliper Technologies, Mountain View, CA, USA), autofluorescence fusion protein Includes substrates (eg, US Pat. No. 6,248,550), and FRET-based assays (Z-LYTE®; Invitrogen, CA, USA). As will be appreciated by those skilled in the art, other active kinases involved in abnormal cell growth can be used to determine the selectivity of a kinase inhibitor for Syk.

In some embodiments, inhibitory compounds used for the treatment of cell proliferative disorders include Syk / Flt-3 kinase inhibitor compounds. “Syk / Flt-3 kinase inhibitor compound” or “Syk / Flt-3 kinase inhibitor” refers to, for example, by directly inhibiting the activity of Flt-3 kinase itself, or by the IC described herein. _It refers to a Syk inhibitor compound that can also inhibit Flt-3 kinase by inhibiting the interaction with other cellular targets required for proper Flt-3 function in the ₅₀ range. Compounds that are Syk / Flt-3 inhibitors are generally about 5 uM or less for Flt-3 kinase activity, eg, the ability of Flt-3 kinase to phosphorylate synthetic or endogenous substrates, in vitro or in cellular assays. A compound that exhibits an IC ₅₀ in the range of about 1 uM or less, about 500 nm or less, about 100 nm or less, about 50 nm or less, about 10 nm or less, or about 1 nm or less. For example, exemplary Syk / Flt-3 inhibitory compounds can be found in the genus of compounds disclosed in US Patent Application No. 10 / 631,029 and PCT Publication No. WO 2004/014382. One skilled in the art will appreciate that compounds that exhibit a lower IC ₅₀ , eg, an IC ₅₀ within a range that is about 100 nM, 10 nM, 1 nM, or even lower, are useful in the methods of the invention.

  A variety of kinase inhibitors can be used in the methods of the invention and are intended to include the salts, hydrates, solvates and N-oxides of the corresponding inhibitor compounds where applicable. In some embodiments, the Syk kinase or Syk / Flt-3 kinase inhibitor is 2 as described in US patent application Ser. No. 10 / 631,029 and published PCT application No. WO 2004/014382. , 4-pyrimidinediamine compounds and various derivatives thereof. These patents are incorporated herein by reference in their entirety. These compounds generally comprise a 2,4-pyrimidinediamine “core” having the following structure and numbering scheme:

該化合物は、Ｃ２窒素（Ｎ２）で置換されて第二級アミンを形成し、場合により次の位置、すなわちＣ４窒素（Ｎ４）、およびＣ５位および／またはＣ６位の１またはそれ以上でさらに置換されている。Ｎ４で置換されている時、置換基は、第二級アミンを形成する。Ｎ２における置換基、ならびに他の位置における任意置換基は、特徴および物理化学的性質において広い範囲にわたることができる。例えば、１または複数のこの置換基は、分枝、直鎖、または環状アルキル、分枝、直鎖、または環状ヘテロアルキル、単環式もしくは多環式アリール、単環式もしくは多環式ヘテロアリール、またはこれらの基の組み合わせであってもよい。これらの置換基は、米国特許出願第１０／６３１，０２９号およびＰＣＴ公報第ＷＯ２００４／０１４３８２号に記載されているように、さらに置換されてもよい。

The compound is substituted with a C2 nitrogen (N2) to form a secondary amine, optionally further substituted at the next position, namely the C4 nitrogen (N4), and one or more of the C5 and / or C6 positions. Has been. When substituted with N4, the substituent forms a secondary amine. The substituents at N2, as well as optional substituents at other positions, can range over a wide range in characteristics and physicochemical properties. For example, one or more of the substituents may be branched, straight chain, or cyclic alkyl, branched, straight chain, or cyclic heteroalkyl, monocyclic or polycyclic aryl, monocyclic or polycyclic heteroaryl Or a combination of these groups. These substituents may be further substituted as described in US Patent Application No. 10 / 631,029 and PCT Publication No. WO 2004/014382.

N2 and / or N4 substituents may be attached directly to their respective nitrogen atoms, or they are spaced from their respective nitrogen atoms via a linker that may be the same or different. It may be. The nature of these linkers can vary widely and can include virtually any combination of atoms or groups useful for spacing one molecular part from each other. For example, the linker may be an acyclic hydrocarbon bridge (eg, saturated or unsaturated alkyleno such as methano, etano, etheno, propano, prop [1] eno, butano, buto [1] eno, buto [2] eno, buta [ 1,3] dieno), monocyclic or polycyclic hydrocarbon bridges (eg, [1,2] benzeno, [2,3] naphthaleno, etc.), simple acyclic heteroatoms or heteroalkyldiyl bridges (eg, For example, —O—, —S—, —S—O—, —NH—, —PH—, —C (O) —, —C (O) NH—, —S (O) —, —S (O _{) 2 -, - S (O} ) NH -, - S (O) 2 NH -, - O-CH 2 -, - CH 2 -O-CH 2 -, - O-CH = CH-CH 2 - , etc.) Monocyclic or polycyclic heteroaryl bridges (eg, [3,4] furano, pyridino, thiopheno, piperi Roh, piperazino, Pirajinino, pyrrolidino, etc.), or it may be a combination of such a bridge.

  The substituents at the N2, N4, C5 and / or C6 positions, and the optional linker may be further substituted with one or more of the same or different substituents. The nature of these substituents may vary widely. Non-limiting examples of suitable substituents include branched, straight chain, or cyclic alkyl, monocyclic or polycyclic aryl, branched, straight chain, or cyclic heteroalkyl, monocyclic or polycyclic heteroaryl, Halo, branched, straight chain, or cyclic haloalkyl, hydroxyl, oxo, thioxo, branched, straight chain, or cyclic alkoxy, branched, straight chain, or cyclic haloalkoxy, trifluoromethoxy, monocyclic or polycyclic Aryloxy, monocyclic or polycyclic heteroaryloxy, ether, alcohol, sulfide, thioether, sulfanyl (thiol), imine, azo, azide, amine (primary, secondary, and tertiary), Nitrile (any isomer), cyanate (any isomer), thiocyanate (any isomer), nitroso, nitro, diazo Sulfoxide, sulfonyl, sulfonic acid, sulfamide, sulfonamide, sulfamine ester, aldehyde, ketone, carboxylic acid, ester, amide, amidine, formazine, amino acid, acetylene, carbamate, lactone, lactam, glucoside, gluconlide, sulfone, ketal, acetal, Includes thioketals, oximes, oxamic acids, oxamine esters, and the like, and combinations of these groups. Substituents bearing reactive functional groups may be protected or unprotected, as is well known in the art.

  Certain embodiments of Syk kinase inhibitor compounds are also described in US patent application Ser. No. 10 / 631,029 filed Jul. 29, 2003; U.S. patent application Ser. No. 10/903, filed Jul. 30, 2004. , 263 (US Application Publication No. 2005/0234049); US Patent Application No. 10 / 903,870 filed July 30, 2004 (US Application Publication No. 2005/0209224); US Patent Application No. 60 / 630,808; and PCT Publication No. WO 2004/014382. Prodrug forms of 2,4-pyrimidinediamine compounds are described in US patent application Ser. No. 11 / 337,049 filed Jan. 19, 2006. All publications and patent applications are incorporated herein by reference in their entirety. These compounds are also disclosed in Appendices A, B, C, and D of US Provisional Application No. 60 / 672,648, filed April 18, 2005. The entire contents of which are hereby incorporated by reference.

  In some embodiments, the Syk inhibitor compound is a U.S. Patent Application No. 60 / 494,008 filed on Aug. 7, 2003, U.S. Patent Application No. 60/572 filed May 18, 2004. , 534; and US Application Publication No. 2005/0113398 (Application No. 10 / 913,270). All patent applications and publications are incorporated herein by reference in their entirety. These compounds are also disclosed in Appendices 1, 2, and 3 of US Provisional Application No. 60 / 672,648, filed April 18, 2005. The entire contents of which are hereby incorporated by reference.

  Exemplary embodiments of Syk or Syk / Flt-3 kinase inhibitors based on 2,4-pyrimidinediamine for the treatment of the conditions and disorders described herein include, inter alia, the following structures:

を有する化合物、およびその様々な塩、水和物、溶媒和物、およびＮ−酸化物を包含する。

And the various salts, hydrates, solvates, and N-oxides thereof.

  Other exemplary embodiments of Syk or Syk / Flt-3 kinase inhibitors based on 2,4-pyrimidinediamine include inter alia N4- (2,2-dimethyl-3-oxo-4H-5-pyrido [ 1,4] oxazin-6-yl) -5-fluoro-N2- (3,4,5-trimethoxyphenyl) -2,4-pyrimidinediamine (Compound VI); N4- (2,2-dimethyl-4 -[(Dihydrogenphosphonoxy) methyl] -3-oxo-5-pyrido [1,4] oxazin-6-yl) -5-fluoro-N2- (3,4,5-trimethoxyphenyl) -2,4 -Pyrimidinediamine (compound VII); and various salts thereof (such as calcium salts), hydrates, solvates, and N-oxides.

  In some embodiments, these Syk kinase inhibitors have a pyridine, pyrimidine, or triazine ring as described in published US Patent Application No. 2004/0106615 and PCT Publication No. WO 2004/016597. A base compound may be included. These are incorporated herein by reference. Generally, the pyridine, pyrimidine, or triazine ring is directly attached to a 6-membered aryl or heteroaryl ring having 0-3 nitrogen atoms.

  In some embodiments, the Syk kinase inhibitor is an amino- or -amino acid as described in PCT Publication Nos. Includes compounds based on diaminotriazole. These are incorporated herein by reference. Aminotriazole compounds typically have a substituent on the nitrogen atom at the 3- or 4-position of the triazole ring, or have an amino substituent on this ring. Exemplary aminotriazoles include, among others, aminotriazole pyridines and aminotriazole pyrimidines (see, eg, WO 2005/013982). Similarly, diaminotriazole compounds that inhibit kinases have multiple substituents on one of the amino groups and one substituent on the nitrogen atom in the 3- or 4-position of the triazole ring. Exemplary kinase inhibitors based on diaminotriazole are described in WO 2004/046120 and US application 2004/0214817.

  In some embodiments, the Syk kinase inhibitor is as described in US Pat. No. 6,849,641, published US patent application No. 2004/0053931, and PCT Publication No. WO 03/000688. And compounds based on azaindole. All of these patents are incorporated herein by reference. US Pat. No. 6,849,641 describes 3-heteroarylideneazaindoline-2-one compounds. Similarly, US Patent Application No. 2004/0053931 and PCT Publication No. WO 03/000688 describe, among other things, aza in which pyrrolopyridine has an aromatic or heterocyclic substituent (eg, benzyl or indolyl) in the 2 or 3 position. Describes indole compounds.

  In other embodiments, the Syk kinase inhibitor comprises a benzimidazole based compound, as described in published US patent application 2004/0048868 and PCT publication WO 03/020698. . Both of these patents are incorporated herein by reference in their entirety. These compounds typically have substituents at the 1- and 2-positions of imidazolyl, with additional substituents on the benzyl ring. Exemplary substituents at the 2-position are aryl or heteroaryl, such as pyrazolyl, triazolyl, imidazolyl, indolyl, indazolyl, thienopyrazolyl, tetrahydroindazolyl, tetrahydrocyclopentapyrazolyl, dihydrofuroprazolyl, oxodihydropyridazinyl, tetrahydro A pyrrolopyrazolyl, oxotetrahydropyrrolopyrazolyl, tetrahydropyranopyrazolyl, tetrahydropyridinopyrazolyl, or oxodihydropyridinopyrazolyl group.

  In some embodiments, Syk kinase inhibitors are disclosed in US Pat. Nos. 6,762,179, published US patent applications 2003/0119856 and 2005/0004152, and PCT Publication No. WO 02/096905. As described, it includes compounds based on thiazole. These patents are incorporated herein by reference. An exemplary thiazole-based inhibitor is 4-thiazolyl pyrimidine in which the pyrimidine has substituents at the 2 and 4 positions. Typically the group at the 2 position is an unsubstituted or substituted amine. Substituents on the amine are generally monocyclic and heterocyclic rings such as substituted phenyl, indanyl, naphthyl, pyrimidinyl, or pyridyl rings.

  In some embodiments, the Syk kinase inhibitor is based on pyrrolopyrimidine as described in published US patent application Nos. 2004/0142947 and PCT publications WO 03/000695 and WO 2004/016597. The following compounds are included. These patents are incorporated herein by reference. In some embodiments, the pyrrolopyrimidine is attached to the 3-position of the indole ring. Generally, this indole has a substituent at the 1-position and / or 5-position. An additional substituent may be present at the 4-position of the pyrrolopyrimidine. This includes, among others, cyano, halo, hydroxy, nitro, aryl, heteroaryl, alkenyl, or alkynyl.

In some embodiments, the Syk kinase inhibitor comprises an indazole-based compound, as described in published US Patent Application No. 2005/0009876 and US Pat. No. 6,534,524. Contains. These patents are incorporated herein by reference in their entirety. US Pat. No. 6,534,524 discloses inhibitory compounds in which indazole has a substituent at the 3 and / or 5 position. Substituents at the 3-position are, among others, unsubstituted aryl or substituted or unsubstituted heteroaryl, or CH═CH—R, or CH═N—R, wherein R is substituted or unsubstituted alkyl, alkenyl, cycloalkyl , Heterocycloalkyl, aryl, or heteroaryl). Substituents at the 5-position are, among others, substituted or unsubstituted aryl, heteroaryl, or Y—X where Y is O, S, C═CH ₂ , C═O, S═O, SO ₂ , alkylidene. , NH, N-alkyl, R ¹ is substituted or unsubstituted aryl, heteroaryl, or N—R ′, where R ′ is alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl Similarly, US Patent Application No. 2005/0009876 discloses compounds in which indazole has a substituent at the 3-position and / or 5-position of the indazole. , Substituted or unsubstituted aryl, or heteroaryl, or heterocycle fused to phenyl is alkyl, such as alkanyl The substituent at the 5-position is, inter alia, halogen, hydroxy, carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, aryl, substituted Aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, substituted or unsubstituted heterocycloalkyl, ester, amide, cyano, or substituted or unsubstituted amine.

  In some embodiments, the Syk kinase inhibitor is as described in US Pat. No. 6,573,295, published US patent application 2002/0062031, and PCT publication WO 00/27802. In addition to bicyclic compounds. All of these patents are incorporated herein by reference. In some of these embodiments, the unsubstituted or substituted benzyl is fused to a cycloalkyl that is also substituted or unsubstituted. An exemplary cycloalkyl is heptenyl. An exemplary bicyclic inhibitor compound is {4- [2- (7-carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo [b] oxepin- (S) -5-ylcarbamoyl) -2-phenylacetylamino-ethyl] -2-phosphono-phenyl} -phosphonic acid; {4-[(S) -2-acetylamino-2- (3-carbamoyl-2-cicyclomethoxymethoxy-6,7, 8,9-tetrahydro-5H-benzocycloheptene- (S) -5-ylcarbamoyl) -ethyl] -phenoxy} -acetic acid; and (4-[(s) -2-acetylamino-2- (3- Carbamoyl-2-cicycloxymethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene- (S) -5-ylcarbamoyl) -ethyl] -2-carboxymethyl Phenoxy - acetic acid.

  In other embodiments, the kinase inhibitor comprises a chromenone oxime compound, as described in published US Patent Application No. 2004/0198750 and PCT Publication No. WO 2004/092154. These patents are incorporated herein by reference. In general, the substituents are at the 2 and 7 positions of the chromenone oxime nucleus.

  Other kinase inhibitor compounds include substituted heterocycles described in PCT Publication No. WO 99/47529 (eg thiazole, oxazole, isoxazole, diazole, oxadiazole, dioxazole, furan, and pyridine), and substituted aryl, Or 5- or 6-membered heteroaryl rings as described in PCT Publication No. WO 2004/085388; and Lai et al., 2003, Bioorg Med Chem Lett. 13 (18): 3sulfonamides as described in 3111-4. Other kinase inhibitory compounds will be apparent to those skilled in the art and can be tested for Syk kinase inhibitory activity using the guidance provided herein.

  The compounds can be tested for their inhibitory effect on Syk and / or Flt-3 kinase in various biochemical and cellular assays. Syk kinase phosphorylates LAT and PLC-γl, which results in degranulation in mast cells and / or basophils, among others. Syk kinase activity is also observed in response to T-cell receptor stimulation. It should be understood that any of these activities can be used to confirm the activity of a Syk inhibitor compound. In some embodiments, the Syk kinase assay is a degranulation assay based on measurement of granule content release following stimulation with anti-IgE. These assays include, for example, measurement of tryptase, histamine, leukotriene LTC4, or hexosaminidase release. In other embodiments, the activity of isolated Syk kinase or an active fragment thereof and an inhibitory compound of a Syk kinase substrate (eg, a synthetic peptide or protein known to be phosphorylated by Syk in a signaling cascade). It is determined by contacting in the presence and assessing whether Syk kinase phosphorylates this substrate. Alternatively, the assay can be performed using cells that express Syk kinase. These cells can express Syk kinase endogenously or they can be designed to express recombinant Syk kinase. These cells can optionally express a Syk kinase substrate. Suitable cells for performing such confirmation assays, as well as methods for designing suitable cells, will be apparent to those skilled in the art. Suitable Syk kinase substrates include, but are not limited to, human band 3 protein (Wang et al., 1999, J Biol Chem. 274 (45), 32159-32166); protein kinase C (Kawakami et al., 2003, Proc Natl Acad Sci USA, 100 (16): 9470-5), tubulin (Peters et al., 1996, J. Biol Chem. 271: 4755), cortactin (Maruyama et al., 1996, J. Biol Chem. 271: 6631), and p50. / HS1 (Ruzene et al., 1996, Biochemistry 35: 1527). Specific examples of biochemical and cellular assays suitable for confirming the activity of Syk inhibitory compounds are described in Fox et al., 1998, Protein Science, 7: 2249, US Patent Application No. 10 / 631,029, WO 2004 / No. 014382 and the references cited therein. All of these patents are incorporated herein by reference.

  Activation of Flt-3 kinase is due to autophosphorylation and, inter alia, of Src homolog 2 (SH2)-, which contains inositol-5-phosphatase (SHIP) and 100-kD protein in monocytic THP-1 cells. Phosphorylation of several cell substrates: phosphorylation of Shc and Cb1 in bone marrow cells; β-arrestin; SH2-containing tyrosine phosphatase, and Cb1-b in pro-B cells (eg Rottapel et al., 1994, Oncogene 9 : 1755-1765; see Zhang et al., 1999, J. Leukoc.Biol.65: 372-380). In some embodiments, the activity of Flt-3 kinase can be measured by the use of antibodies to phosphorylated forms of Flt-3 kinase (ie autophosphorylation assay; Kiyoi et al., 1998, Leukemia 12: 1333). -1337). In some embodiments, the activity is such that the isolated Flt-3 kinase or an active fragment thereof and an inhibitory compound are phosphorylated by a Flt-3 kinase substrate (eg, Flt-3 in a signaling cascade). Can be determined by contacting in the presence of a known synthetic peptide or protein. Alternatively, the assay can be performed using cells that endogenously express Flt-3 kinase or that are designed to express recombinant Flt-3 kinase (eg, Yamamoto et al., 2001). , Blood 97 (8): 2434-2439). These cells can optionally express a Flt-3 kinase substrate. In some embodiments, the Flt-3 expressing cells are a variety of downstream targets as described above, eg, phospholipase C- (PLC), p85 subunit of phosphatidylinositol 3′-kinase (PI3K), SHC, SHP. -2, SHIP, GRB2, VAV, Fyn kinase, Src kinase, Stat5 signaling protein, and activation of ERK.

  Any number of in vitro and in vivo assays may be used to determine the effect of these inhibitory compounds on cell proliferation. For example, proliferating cells can be appropriately cultured in vitro and treated with the compound. The ability to grow in a cell population is determined by dye staining (eg trypan blue dye-exclusion; 3-4,5-dimethylthiazole-2,5-diphenyltetrazolium (MTT); and annexin V), or cell sorting technology (eg propidium iodide). Fluorescence activated cell sorting with id). In vivo assays for cell proliferation may be based on transplantation of tumor cells into experimental animals followed by administration of these inhibitory compounds. These and other methods of assessing cell proliferation will be apparent to those skilled in the art.

6.3 Dosage One or more active compounds, or compositions thereof, can be used in an amount effective to treat or prevent the particular disease being treated. The compounds can be administered therapeutically to obtain a therapeutic effect or prophylactically to obtain a prophylactic effect. A therapeutic effect is one or more of the symptoms associated with the underlying cell proliferative disorder being treated, such as the eradication or amelioration of a lymphoid neoplasm, bone marrow neoplasm, virus-related tumor, and / or the underlying disorder. Meaning further eradication or improvement, so patients are still reporting improved status despite the patient still suffering from the underlying disorder. The therapeutic effect also includes stopping or delaying the progression of the disease, regardless of whether improvement is perceived.

  For prophylactic administration, the active compounds are characterized by abnormal cell proliferation, or the risk of developing or associated with disorders associated therewith, such as the various disorders already described above. Can be administered to certain patients. For example, if a patient is diagnosed with a tumor but there are no signs of metastasis, these inhibitory compounds can be administered prophylactically to inhibit tumor metastasis.

  The amount of one or more inhibitory compounds administered will depend on a variety of factors. This includes, for example, the particular indication being treated, the mode of administration, whether the desired effect is prophylactic or therapeutic, the severity of the indication being treated, and the age and weight of the patient, the particular activity Includes bioavailability of compounds. The determination of an effective dosage is well within the ability of those skilled in the art.

Initial dosage can be estimated initially from in vitro assays. For example, the initial dosage for use in animals is to obtain a circulating blood or serum concentration of a compound that inhibits Syk or Flt-3 sufficient to reduce cell proliferation or tumor cell invasiveness. Can be blended. Alternatively, the initial dosage for animal use is to obtain a circulating blood or serum concentration of the active compound that is equal to or greater than the IC ₅₀ as measured in a Syk kinase or Flt-3 inhibition assay. Can be blended. Taking into account the bioavailability of a particular inhibitory compound, the calculation of dosages to obtain such circulating blood or serum concentrations is well within the ability of those skilled in the art. For example, the reader may read The Pharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46, 1975, in Fingl and Woodbury, “General Principles”, and references cited therein. Initial dosages can also be estimated from in vivo data, such as animal models. Animal models useful for testing the effectiveness of compounds for treating or preventing diseases characterized by, or caused by, Syk kinase or Flt-3 kinase activity are It is described in the specification.

  The dosage is typically within the range of about 1 mg / kg / day to about 100 mg / kg / day, 200 mg / kg / day, 300 mg / kg / day, 400 mg / kg / day, or 500 mg / kg / day. There may be higher or lower depending on the activity of the inhibitory compound, its bioavailability, mode of administration, and various factors discussed above, among other factors. Dosage amount and interval can be adjusted individually to provide plasma levels of one or more of the active compounds which are sufficient to maintain therapeutic or prophylactic effect. In cases of local administration or selective uptake, eg local local administration, the effective local concentration of the active compound may not be related to plasma concentration. One skilled in the art will be able to optimize the effective dosage without undue experimentation.

  The compound may be administered once a day, 2-3 times or several times a day, or even many times per day, depending on, inter alia, the indication being treated and the judgment of the prescribing physician. it can.

  Preferably, the active compound will provide a therapeutic or prophylactic effect without causing substantial toxicity. The toxicity of the active compound can be determined using standard pharmaceutical procedures. The dose ratio between toxic and therapeutic (or prophylactic) effect is the therapeutic index. Active compounds that exhibit high therapeutic indices are preferred.

6.4 Administration When used to treat or prevent cell proliferative disorders, the Syk or Syk / Flt-3 inhibitory compound may be used alone or as a mixture of one or more active compounds or such diseases And / or can be administered in admixture with or in combination with other agents useful in the treatment of symptoms associated with such diseases. These active compounds can be administered per se or as a pharmaceutical composition.

  Pharmaceutical compositions containing the active compounds of the present invention can be manufactured using conventional mixing, dissolving, granulating, sugar-coated tableting, emulsifying, encapsulating, entrapping, or lyophilizing methods. The composition is one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate processing of the active compound into a pharmaceutically usable preparation. Can be blended in a conventional manner. The actual pharmaceutical composition administered will depend on the method of administration. Nearly any method of administration can be used, including, for example, topical, oral, systemic, inhalation, infusion, transdermal, and the like.

  The active compound can be incorporated into the pharmaceutical composition by itself or in the form of a pharmaceutically acceptable salt. As used herein, the expression “pharmaceutically acceptable salt” substantially retains the biological effectiveness and properties of the active compound and is also biologically or otherwise. Means a salt that is not undesirable. Such salts can be prepared from inorganic and organic acids and bases as is well known in the art. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases.

  For topical administration, the active compounds can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well known in the art.

  Systemic formulations are designed for administration by injection, for example subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, as well as for transdermal, transmucosal oral, or pulmonary administration. Is included.

  Useful injectable preparations include sterile suspensions, solutions or emulsions of one or more of the active compounds in an aqueous or oily vehicle. These compositions may also contain formulating agents such as suspensions, stabilizers, and / or dispersants. These formulations for injection may be presented in unit dosage form, for example in ampoules, or in multi-dose containers, and may contain added preservatives.

  Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle prior to use, including but not limited to sterile pyrogen-free water, buffering agents, dextrose solutions, and the like. Good. For this purpose, the active compounds can be dried by any technique known in the art, such as lyophilization, and reconstituted prior to use.

  For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

  For oral administration, the pharmaceutical composition may be, for example, by conventional means, a pharmaceutically acceptable excipient, such as a binder (eg, pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); Agents (eg, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (eg, magnesium stearate, talc, or silica); disintegrants (eg, potato starch, or sodium starch glycolate); or wetting agents (eg, It may take the form of tablets or capsules prepared with sodium lauryl sulfate. The tablets may be coated with methods known in the art, such as sugar or enteric coatings.

  Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or these may be dried for constitution with water or other suitable vehicle prior to use. It may be presented as a substance. Such liquid preparations are prepared by conventional means with pharmaceutically acceptable additives such as suspensions (eg sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (eg lecithin or acacia); Aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oil); and preservatives (eg, methyl or propyl-p-hydroxybenzoate, or sorbic acid) can be used. These preparations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents as desired. Preparations for oral administration can be suitably formulated to give controlled release of the active compound.

  For buccal administration, the composition may take the form of tablets or lozenges formulated in conventional manner.

  For the rectal and vaginal routes of administration, the active compounds may be formulated as traditional suppository bases, for example cocoa butter or other glyceride containing solutions (retention enema) suppositories or ointments.

  For administration by inhalation, the active compounds are in the form of aerosol sprays from pressurized packs or nebulizers, suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or others Can be conveniently delivered using any suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

  For extended delivery, the active compounds may be formulated as a depot preparation for administration by implantation, eg, subcutaneous, intradermal, or intramuscular injection. Thus, for example, the active ingredient can be obtained using a suitable polymer or hydrophobic material (eg as an acceptable emulsion in oil), or as an ion exchange resin, or as a slightly insoluble derivative; You may mix | blend as a salt without property.

  Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch that slowly releases the active compound for percutaneous absorption can be used. For this purpose, permeation enhancers can be used to facilitate transdermal penetration of the active compound. Suitable transdermal patches are, for example, US Pat. No. 5,407,713; US Pat. No. 5,352,456; US Pat. No. 5,332,213; US Pat. No. 5,336,168; US Pat. No. 5,290,561; US Pat. No. 5,254,346; US Pat. No. 5,164,189; US Pat. No. 5,163,899; US Pat. No. 5,088,977; No. 5,087,240; US Pat. No. 5,008,110; and US Pat. No. 4,921,475.

  Alternatively, other pharmaceutical delivery systems can be used. Liposomes and emulsions are well known examples of delivery vehicles that can be used to deliver active compounds. Certain organic solvents, such as dimethyl sulfoxide (DMSO), can also be used at the cost of usually greater toxicity.

  The pharmaceutical compositions may be presented in packs or dispensers that may contain one or more unit dosage forms containing the active compound, if desired. This pack may for example comprise a metal or plastic foil, for example a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

7). Example 7.1 Example 1: Effect of Syk Inhibiting Compound on Syk-Induced Pre-B Cell Transformation Experiments demonstrating inhibition of growth of Syk-transformed cells by a Syk inhibiting compound are described in Wossning, T .; Herzog, S .; Kohler, F .; Meixlsperger, S .; Kulathu, Y .; Mittler, G .; Abe, A .; , And Jumaa, H .; , “The protein tyrosine kinase Sym is involved in transformation transformation and leukemia development” (preparing manuscript). The experiments described in this manuscript are briefly summarized below.

  Retrovirally transducing a newly isolated bone marrow pre-B cell carrying an IRES-GFP vector expressing Tel-Syk with an abnormal Syk mutant resulting from chromosomal translocation Tel-Syk Were tested for their ability to transform pre-B cells. Recovery of IL-7 from these cell cultures results in the enrichment of Tel-Syk expressing cells and the generation of stable IL-7 independent cell lines, indicating that Tel-Syk expression is pre-B cells It can be shown that the growth and transformation of can be promoted. Transformation of pre-B cells with Tel-Syk activity was confirmed by injecting Tel-SYK transduced cells into RAG / γC − / − mice. This resulted in animals showing symptoms of myelodysplastic disease. The data show that Tel-Syk expressing cells can proliferate rapidly in vivo, thereby inducing aggressive leukemia.

  To test whether inhibition of Syk activity in cells transformed with Tel-Syk interferes with cell proliferation, the effect of the Syk kinase inhibitor 2,4-pyrimidinediamine compound IV was determined on the DNA content of treated cells. Tested in vitro by examining the amount. Compound IV effectively prevented Syk-induced pre-B cell proliferation and allowed the pre-B cells to differentiate, as shown by the expression of the kappa light chain in these cells. The data shows that Syk expression may be required for the growth of leukemia cells, and that preventing Syk kinase activity can result in inhibition of leukemia cell growth.

7.2 Example 2: Effect of Syk inhibitor on established leukemia To determine the effect of Syk inhibitor on the proliferation of leukemia cells, several tumorigenic pre-Bs that induce leukemia after injection into mice A cell line was used. Since tumorigenic cells require the pre-B cell receptor (pre-BCR) for growth and Syk is an important protein for the pre-BCR signaling cascade, Syk inhibitors are Are expected to inhibit the growth of tumorigenic pre-B cells. In one experiment, the ability of Compound VI to inhibit proliferation was examined using pre-B cells transduced with a Myc expression vector and cultured in the absence of IL-7. To examine their in vivo growth capacity, cells were injected into RAG / γC − + / − mice. The ability of VI to prevent proliferation was tested in vitro using the Myc transduced cell line (FIGS. 2A and 2B).

The ability of VI to prevent proliferation was also examined using a tumorigenic SLP-65 ^{− / −} pre-B cell line. To examine their in vivo growth capacity, cells were injected into RAG / γC − + / − mice. The ability of VI to prevent the growth of oncogenic SLP-65 ^{− / −} pre-B cell lines was tested in vitro (FIGS. 3A and 3B).

  Other leukemia cell lines useful for examining the effects of Syk inhibitor compounds include, but are not limited to, the B cell lymphoid cell line JM1 (ATCC No. CRL-10423), the Hodgkin lymphoma cell line RPMI-6666 ( ATCC No. CCL-113), promyelocytic leukemia cell line HL-60 clone 15 (ATCC No. CRL-1964), Burkitt lymphoma cell line GL-10 (ATCC No. CRL-2392), acute myeloid Leukemia cell line BDCM (ATCC No. CRL-2740), myelomonocytic leukemia (EBV) cell line CESS (ATCC No. TIB-190), EBV transformed plasmacytoma / myeloma cell line MC-CAR (ATCC No. CRL-8083), and EBV transformed plasma cell leukemia cell line ARH-77 (AT It encompasses C No.CRL-1621).

7.3 Example 3: In vivo evaluation of the calcium salt form of Compound VII and in a MV4-11 acute myeloid leukemia (AML) intravenous tumor implantation model in NOD-SCID immunodeficient mice pre-treated with cyclophosphamide Cell Titration Flt-3 ITD mutations were associated with poor prognosis and reduced remission rates in affected AML patients and made them attractive targets for therapeutic intervention. The study shows that mutant Flt-3 ITD receptor dimerizes Flt-3 ligand-independently, and receptor autophosphorylation results in constitutive activation, increased proliferation, and mutant cells It has been shown that this results in growth factor independence. Initial studies have shown that 5 million MV4-11 human acute myeloid leukemia cells with the Flt-3 ITD mutation are intravenous (iv) when calcium salt form VII reduces subcutaneous tumor growth, prolongs survival. .) It was shown that the amount of whole body tumor tissue of the inoculated mice was reduced. This study showed that i.v. of 5 million or 10 million MV4-11 leukemia cells into NOD-SCID mice pre-treated with cyclophosphamide. v. Performed to demonstrate the efficacy of VII administration in inhibiting tumor growth through oral treatment twice daily at 40 mg / kg VII for disease progression, severity, and survival resulting from infusion did.

7.3.1 Experimental Method Cell Lines Leukemia cell line: MV4-11 human acute myeloid leukemia (AML) (supplier: American Type Culture Collection (ATCC)) was maintained and collected by Oncology Group at Rigel. The cells were tested for IMPACT on November 30, 2004 and were subjected to ectromelia, EDIM, Hantaan, K virus, LCMV, LDEV, MAD, mCMV, MHV, MMV, MPV, MTV, mycoplasma sp. , Polyoma, PVM, REO3, Sendai, TMEV, and GDVII.

Cell line maintenance: MV4-11 cells were placed in Iskov modified Dulbecco's medium (ATCC® number: 30-2005), 1.5 g / L sodium bicarbonate, 80%; fetal calf serum, 20% (GIBCO BRL, Carlsbad, CA) maintained with 4 mM L-glutamine adjusted to contain 1% penicillin / streptomycin (10,000 IU / ml and 10,000 ug / ml) (CellGro / Media Tech, catalog number 30-002-Cl) To do. Cells were harvested in logarithmic growth phase, washed and poured into fresh medium. MV4-11 cells were seeded at 5 × 10 ⁶ or 10 × 10 ⁶ cells per animal.

Pharmacology Details of the test products and test formulations used in this study are listed in the following formulation sheet. Test products and vehicle formulations were made by the drug department prior to the start of dosing (vehicle (lot: 1024-15-01); 8 mg / mL VII calcium salt (lot: 1024-15-03). Calcium salt form VII Were prepared at equal concentrations of VI.The formulations were stored at 4 ° C., photoprotected, and vortexed prior to use.

動物および収容
生後約１０週のメスＮＯＤ．ＣＢ１７−Ｐｒｋｄｃ ｓｃｉｄ／Ｊ（ＮＯＤ／ＳＣＩＤ）マウス（ｎ＝６０；出生日２００５年５月３０日；Ｊａｃｋｓｏｎ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｂａｒ Ｈａｒｂｏｒ，ＭＥから２００５年７月１９日に入荷）を調査のために用いた。ＮＯＤ−ＳＣＩＤマウス（系統番号００１３０３）は、Ｊａｃｋｓｏｎ Ｌａｂｏｒａｔｏｒｙからのみ入手可能であり、ＮＯＤ−ＳＣＩＤマウスに独特な生来の適応的免疫における多重欠陥は、ヒト造血細胞での再構成のための優れたインビボ環境を提供する。約５０匹のマウスにシクロホスファミドを与え、この調査の生存中部分のために用いたが、追加のマウスは、植付けレベルのフローサイトメトリー分析用のバックグラウンド染色の評価のための非腫瘍対照のために用いた。わずかに年長のマウス（＞１９グラム）を、シクロホスファミド注入後の過剰な体重減少による早期の死を避けようとするために用いた（Ｖ０５０１４４と比較して）。

Animals and housing Female NOD. CB17-Prkdc scid / J (NOD / SCID) mice (n = 60; date of birth May 30, 2005; received from Jackson Laboratories, Bar Harbor, ME on July 19, 2005) were used for the study. . NOD-SCID mice (strain # 001303) are only available from Jackson Laboratory, and multiple defects in innate adaptive immunity unique to NOD-SCID mice are excellent in vivo for reconstitution with human hematopoietic cells. Provide an environment. Approximately 50 mice were given cyclophosphamide and used for the live part of this study, but additional mice were non-tumor for evaluation of background staining for planting level flow cytometry analysis Used for control. Slightly older mice (> 19 grams) were used to try to avoid premature death due to excessive weight loss after cyclophosphamide injection (compared to V050144).

  Animals were housed 5 per cage in a Microisolator cage in a ventilated HEPA-filter cage rack (Alternative Design, Siloam Springs, AZ). On arrival, they were acclimated for at least 4 working days before use. Temperature was maintained at 72 ± 5 ° F., relative humidity at 35-70%, and a 12 hour light / dark cycle was used. Mice were given an unlimited number of certified rodent diets (Deans Feed, San Carlos, CA). Autoclaved R.P. O. Unlimited drinking water was available. Cages and food were autoclaved before use. All survival procedures were approved by Rigel IACUC.

  NOD-SCID mice are neither insulitis nor diabetic for the duration of survival, despite the background of NOD autoimmune diabetic tendency, but under conditions that do not contain specific pathogens, Has a high incidence of thymic lymphoma, limiting life expectancy to only 8.5 months. The Emv-30 provirus on chromosome 11 results in a high frequency of thymoma starting at 5-6 months of age in NOD-SCID (3,4), (5), which is the NOD-SCID as recipient. Limit the duration of experiments performed with mice. In this experiment, all surviving mice were terminated when they reached approximately 5 months of age (day 83 of the study) to limit the effect of natural thymoma on outcome variables in this study.

Animal Procedure Bone marrow depletion for enhanced planting: At the end of the acclimatization period for these animals (> 3 weeks in this study), mice were treated for 2 days followed by leukemia via the lateral tail vein as described below. Prepared in 0.9% sterile saline (Sigma, St Louis, MO) by intraperitoneal injection at 150 mg / kg / day for 72 hours remaining time prior to intravenous (iv) injection of cells , Pre-treated with sterile filtered cyclophosphamide (using 0.22 μM syringe filter).

  Tumor inoculation: On day 0 of the study, mice were inoculated with 5 million or 10 million human MV4-11 leukemia cells resuspended in 100 microliters of fresh medium as outlined in Table 1. It was inoculated once by intravenous tail vein injection.

  Dosing: After inoculating mice with human MV4-11 cells (day 0), dosing began on day 17 of the study. Mice were dosed orally (PO) twice daily (bid) with either vehicle or 40 mg / kg VII at 5 mL / kg dosing volume with a minimum 8 hour interval between doses during the study. .

Experimental procedure Tumor model: This study involved tumor implantation in a murine intravenous MV4-11 leukemia model using NOD-SCID immunodeficient mice as a host with cyclophosphamide pretreatment to enhance tumor implantation. It was designed to evaluate the effect of various doses of VII on disease progression, severity, and overall survival. Historical data show that bone marrow is in spite of> 20% weight loss, moribundity, significant loss of motility due to hind limb paralysis, or uniform lethality of tumor cell injection determined by unexpected death as an endpoint. (BM) and variable tumor cell implantation in peripheral blood mononuclear cells (PBMC) are shown (Studies V050123 and V050144). In addition, cyclophosphamide has proven to be necessary for lethality in this model. A published study showing a median survival time of 51 days in vehicle control mice also demonstrates variable BM planting at the expense of a range of 2-19% human cells detected in the BM compartment.

  Although this study was conducted in an attempt to confirm previous efficacy data from the study, similarly, did 5 million or 10 million cell inoculations produce similar discrepancies in tumor cell transplantation in bone marrow and peripheral blood? It was also performed to determine whether or not to modify the resulting mortality in these mice. Detectable levels of planting in some animals are observed as early as 4 weeks after tumor cell injection in pilot studies, and historical efficacy data are determined by this regimen, thus starting approximately 2 weeks after tumor cell injection However, animals were orally (PO) treated with vehicle or R945788 calcium salt at 40 mg / kg on a twice daily (bid) schedule. Animals continued this regimen until euthanized or until the end of the study. In this experiment, all surviving mice were terminated when they reached approximately 5 months of age (day 83 of the study) in order to limit the effects of spontaneous thymoma development. It can be seen that the onset of natural thymoma begins about 5-6 months after birth in NOD-SCID mice.

  Study design: On day 0 (August 12, 2005), mice were challenged i.v. with human MV4-11 cells in fresh medium at 5 million or 10 million cells per mouse via the lateral tail vein. v. Injected. By day 3 after MV4-11 cell injection (August 15, 2005), one mouse was removed from excessive weight loss, potentially by cyclophosphamide treatment, prior to randomization or treatment. Excluded by early death. The remaining animals were randomly placed into treatment groups by body weight and dosing began on day 17 (August 29, 2005) after cell injection. The average body weights for Groups 1-4 were 20.66 ± 0.43, 20.23 ± 0.41, 20.18 ± 0.54, and 20.88 ± 0.59, respectively. Table 1 shows an overview of processing groups.

疾病の進行および疾病の重症度：全体的な疾病の重症度は、体重減少、臨床的観察事項、検死発見事項、および選択された組織中の腫瘍細胞植付けの定量の組み合わせから決定した。マウスを毎週２〜３回体重測定し、パーセント体重変化を計算した。ＩＡＣＵＣ Ｐｒｏｔｏｃｏｌ Ｒｉｇｅｌ ６−２００２，“Ｅｆｆｉｃａｃｙ ｏｆ Ｎｏｖｅｌ Ｃｈｅｍｏｔｈｅｒａｐｅｕｔｉｃｓ ｉｎ Ｈｕｍａｎ／Ｍｏｕｓｅ Ｔｕｍｏｒ Ｘｅｎｏｇｒａｆｔ Ｍｏｄｅｌｓ”に概要が示されているように、嗜眠、かき乱された毛などをともなう、病的状態、明らかな消耗を有する後肢麻痺、重症の悪液質、または２０％超の体重減少の最初の兆候があったとき、動物を調査から除去し、安楽死させた。犠牲時、動物をＣＯ_２ガスで麻酔し、血液を心臓穿刺によって収集した。動物からの血液を、Ｋ２ＥＤＴＡコーティングしたＭｉｃｒｏｔａｉｎｅｒ（ラベンダートップ）チューブに移した。全血を間接的に氷に載せ、以下のセクションに記載されているように、フローサイトメトリー染色のために加工処理した。病気の重症度をさらに評価するために、全体試験において、触知可能なまたは疑わしい腫瘍の位置および描写を検死の際に注意深く書き留めた。

Disease progression and disease severity: The overall disease severity was determined from a combination of weight loss, clinical observations, autopsy findings, and quantification of tumor cell implantation in selected tissues. Mice were weighed 2-3 times weekly and percent body weight change was calculated. IACUC Protocol Rigel 6-2002, “Efficacy of Novel Chemotherapeutics in Human / Mouse Tumor Xenograft Models”, with limbs that are debilitating, with disturbed hair, etc., as outlined in IACUC Protocol Rigel 6-2002, “Efficacy of Novel Chemotherapeutics in Human / Mouse Tumor Xenograft Models” Animals were removed from the study and euthanized when there were first signs of paralysis, severe cachexia, or weight loss greater than 20%. At sacrifice, the animals were anesthetized with CO ₂ gas and blood was collected by cardiac puncture. Blood from the animals was transferred to Microtainer (lavender top) tubes coated with K2EDTA. Whole blood was indirectly placed on ice and processed for flow cytometric staining as described in the following section. To further assess disease severity, palpable or suspicious tumor locations and depictions were carefully noted at necropsy in the overall study.

  Tumor Cell Implantation: To assess systemic tumor tissue volume, the distribution of MV4-11 tumor cells in bone marrow and peripheral blood was analyzed by flow cytometry analysis using several human and mouse surface marker stains. Determined on mouse sacrifice.

  The extent of tumor implantation was evaluated in several mice by isolation of the femur and / or tibia at sacrifice. Bone marrow (BW) was aspirated into a clean Eppendorf tube with 1 ml cold PBS and kept on ice prior to flow staining. All residual tissues or tumors were fixed in 10% buffered formalin for histopathology and immunohistochemistry (IHC). Peripheral blood was also obtained via cardiac puncture and placed in K2EDTA-coated Microtainer (purple) tubes. Only mononuclear cells (PBMC) remained for flow cytometry staining and analysis to lyse erythrocytes, wash samples, and determine tumor cell implantation levels.

  In order to identify human MV4-11 tumor cells, BM and PBMC samples were stained with antibodies specific for mouse CD45, human CD45, human HLA, and human CD33 cell surface markers. This multi-marker strategy was then used to identify and quantify the percentage of human MV4-11 leukemia cells present in randomly selected samples from VII- and vehicle-treated animals. (Appendix 5 and 6). The percentage of human MV4-11 cells was defined as the number of CD33 + HLA + events from all events (defined as the sum of human CD33 + HLA + and murine CD45 + events). Every effort was made to collect and analyze all animals in the study. However, due to the sudden nature of death in vehicle-treated mice in this model, fewer samples were available for analysis and the number of animals was variable between these groups. See Appendix 5 and 6 for details of cell preparation and flow staining and analysis procedures.

  Statistical analysis: Survival of mice was determined by Kaplan-Meier aggressive limit as a determination of relative disease severity (n = 18-20 / group) using the GraphPad Prism 4.0® software package. evaluated. Additional survival curve analysis, including the Log Rank (Mantel-Haenszel) test (two-tailed p-value) for curve comparison, hazard ratio for survival kinetics, and overall median survival, if possible, Calculations were made using Prism 4.0®. All animals surviving at the end of the study (day 83) were reviewed for statistical purposes in the Prism® analysis. Hazard statistics, defined as the slope of the survival curve, are a measure of how quickly a subject is dying. The reported hazard ratio is therefore a comparison of mortality between the two groups. For example, if the hazard ratio is 2.0, the mortality rate in one group is twice that of the other group. In general, Prism 4.0 (registered trademark) G. Standard calculations are used as described in detail in Altman, Practical Statistical for Medical Research, 1991, Chapman and Hall. All results are expressed as mean ± SEM unless otherwise indicated.

In addition, the percent increase in life (% ILS) was determined using the calculated median death date for each group compared to the vehicle control group using the following equation: Where DOD is defined as the date of death:
% ILS = (median DOD _treatment− median DOD _vehicle ) / median DOD _vehicle × 100
Mice treated with 40 mg / kg VII had an unspecified median survival time because an inappropriate number of mice (<50%) died by the end of the study. Therefore, the% ILS calculation assumed a maximum median survival rate of 80 days for this group, meaning it was greater than the calculated value.

  Planting data was analyzed using one-way ANOVA when inequality variance was not detected by Bartlett's test. In this case, an unpaired two-sided student test was performed using Welch correction to account for unequal variances. Normalization of percent CD33 + HLA + cells per total number of cells was performed. Average percent tumor cells were determined by using the following equation for each sample and then measuring the average of these values. The total number of mouse cells was determined using the single cell surface marker murine CD45 and the total number of human events was determined using the dual human surface markers CD33 and HLA.

Percent tumor cells = [(# CD33 + HLA + double positive event) / (total #murine CD45 + and # CD33 + HLA + event)] × 100
Due to the suddenness of death in vehicle-treated mice in this model, fewer samples were available for analysis, limiting our ability to obtain statistical significance. Adequate sample size and random sampling were obtained for VII treated mice. Therefore, comparisons were made between 20, 40, and 80 mg / kg VII treated mice and statistics were performed as described above.

  For categorical variables, such as the total number of tumor animals per group, p-values were also used with a two-sided Fisher exact test with 95% confidence intervals, performed using the GraphPad Prism 4.0® software package. This reflects the results of the treatment group vs. vehicle group comparison.

7.3.2 Results Effect of Compound VII treatment on survival: Overall, animals treated with VII show long survival and reduced number of palpable tumor masses when examined at necropsy It was. Vehicle treated mice inoculated with 5 million MV4-11 cells had a median survival time of 54 days compared to 79 days in 40 mg / kg VII treated mice. Vehicle-treated mice inoculated with 10 million tumors had a median survival time of 54 days, and for mice treated with 40 mg / kg VII, the median death date was an indeterminate number (> 83). Mice treated with 40 mg / kg VII in the 10 million MV4-11 cell group had an indeterminate median survival time, because an inappropriate number of mice (<50%) were Because he died. Therefore, the% ILS calculation assumed a maximum median survival rate of 83 days for this group, meaning it was greater than the calculated value. Significant differences were observed in overall survival between VII-treated and vehicle-treated animals (LogRank p <0.0022 and% ILS = 40 mg / kg VII at 5 million cells). 45% LogRank p <0.0001 and% ILS => 54% for 40 mg / kg VII at 10 million cells (assuming median DOD = 83 for this group)).

８３日目には、ビヒクル処理グループには生存動物はいなかった。これに対して、５百万および１千万ＭＶ４−１１細胞が注入されたＶＩＩ処理グループからのマウスは、それぞれ５匹および９匹の生存マウスを有した。５０％以上のマウスが、ビヒクル対照の０％生存率と比較して、４０ｍｇ／ｋｇでＶＩＩ処理された１千万細胞グループにおいて８３日目まで生存した。

On day 83, there were no surviving animals in the vehicle-treated group. In contrast, mice from the VII treatment group injected with 5 million and 10 million MV4-11 cells had 5 and 9 surviving mice, respectively. More than 50% of the mice survived to day 83 in the 10 million cell group treated with VII at 40 mg / kg compared to 0% survival of the vehicle control.

Effect of Compound VII on Disease Severity and Tumor Frequency and Distribution Significant phenotypic differences were evident when comparing disease progression in vehicle and VII treated animals. At necropsy, palpable tumors in vehicle-treated animals have various lymph nodes (LN), the lower spinal cord near the inferior vena cava (possibly LN), the chest wall, and around the ribs and sternum In the vicinity of the trachea, it was observed in the jaw and throat area (probably salivary gland and LN related). Tumors were also found in the kidneys, ovaries, and heart. In addition, very large tumors were often found around the shoulder, arm, and leg bones. Some mice are noted to have a soft and smooth brain or overt tumor, and one mouse has tumors at necropsy as do some mice from the MOLM13 leukemia model in earlier studies. It seemed to have a very dilated eye.

  Vehicle treated animals clearly showed hind limb paralysis as early as 42 days after MV4-11 infusion in the 5 million cell group and 48 days in the 10 million cell group. At necropsy, palpable tumors in vehicle-treated animals were observed at similar locations as in previous studies. In total, 8 out of 8 mice inoculated with 5 million tumor cells had palpable tumor masses at necropsy, and many mice were identified as having tumors It had multiple anatomical sites. Five mice from the vehicle treatment group were found dead, and four of these mice had no autopsy reports due to advanced decay, resulting in all in this group Only eight autopsies were made. By the end of the study (83 days after MV4-11 transplantation), 12 of 12 vehicle-treated animals were terminated and 5 of these animals were found dead, Three were moribund at the time of the examination, two were sacrificed with> 20% weight loss and two were sacrificed for HLP associated with severe weakness.

  Similarly, 11 out of 12 mice had visible tumor masses and many had multiple sites identified in vehicle treated mice inoculated with 10 million cells. A total of one vehicle-treated animal was found dead and one did not perform an autopsy due to advanced disintegration, resulting in a total of 11 autopsies in this group. At the end of the study (83 days after MV4-11 transplantation), 13 of 13 vehicle-treated mice were terminated and one of these animals was found dead, 3 of these The animals were moribund when examined, 7 had a weight loss of> 20%, and 2 were sacrificed for HLP associated with severe weakness.

  As a good control, mice treated with 40 mg / kg VII show very little tumor, only 3 out of a total of 11 animals necropsied in the 5 million cell group and 13 in the 10 million cell group. Seven of the animals showed some visible or palpable tumor. Most of these animals in the treatment group showed much smaller tumors than the vehicle treatment group. Tumors are located at similar anatomical sites and by the end of the study, 7 out of 12 animals were terminated in a 5 million cell 40 mg / kg VII treatment group and 2 of these animals One was found dead, one was moribund when examined, four had> 20% weight loss and no animals were sacrificed for HLP. Similarly, 40 mg / kg VII treated animals inoculated with 10 million MV4-11 cells showed few tumors and some mice had dilated salivary glands, or slightly dilated LN. At the end of the study, only 4 out of 13 40 mg / kg VII treated mice were terminated so far, 3 mice had> 20% weight loss and 1 mouse was examined It was discovered that he was moribund. Overall, VII-treated mice showed a significant reduction in the total number of tumors, the relative size of the tumor observed at necropsy, and the overall severity of the disease in this study, with 40 mg / kg VII treatment being the initial The effectiveness was similar to that seen in the survey.

  Effect of Compound VII on MV4-11 tumor cell implantation in bone marrow (BM) and peripheral blood (PB): To correlate with previous studies, the level of MV4-11 implantation in bone marrow or peripheral blood was also The animals were examined at termination. Despite our best efforts, some samples were not available for analysis due to the sudden death of vehicle-treated mice in this experiment. Unlike earlier studies, a statistically significant decrease in mean percent MV4-11 tumor cells in the bone marrow and peripheral blood of VII-treated animals at sacrifice was compared to inoculated cells when compared to vehicle-treated animals. Were observed independently of the titration of (Table 3).

  Vehicle treated mice inoculated with 5 million cells had 25% and 61% CD33 + / HLA + human tumor cells in BM (n = 6) and PB (n = 4), respectively. In contrast, VII-treated mice inoculated with 5 million cells have 1% and 0.6% CD33 + / HLA + human tumor cells in BM (n = 8) and PB (n = 8), respectively. (For vehicle control, 40 mg / kg, BM is p = 0.13, PB is p = 0.02). The 10 million cell vehicle treatment group had 13% and 45% CD33 + / HLA + human tumor cells in BM (n = 8) and PB (n = 8), respectively. In contrast, the 10 million cell VII treatment group had 5% and 3% CD33 + / HLA + human tumor cells in BM (n = 13) and PB (n = 11), respectively (vs. vehicle control) Thus, for 40 mg / kg, BM is p = 0.05 and PB is p = 0.007).

（）内の数字は、汚染が疑われる２つのＰＢ試料を除外するために調節された値を反映する（各グループにおいてｎ＝１）。これらの試料は、心臓穿刺による血液収集を試みている間に、乳白色の浸出液を有することが注目され、その後、検死の際に、心臓または胸腺の近くに腫瘍を有することが注目された。これら２つの疑われた値は、ビヒクルおよび４０ｍｇ／ｋｇ ＶＩＩグループ（１千万細胞）において、それぞれ９８％および９３％腫瘍細胞を示した。

Numbers in parentheses reflect values adjusted to exclude two PB samples suspected of contamination (n = 1 in each group). These samples were noted to have a milky white exudate while attempting to collect blood by cardiac puncture, and thereafter at the time of necropsy to have a tumor near the heart or thymus. These two suspected values showed 98% and 93% tumor cells in the vehicle and 40 mg / kg VII group (10 million cells), respectively.

  * Average% tumor cells are defined as CD33 + HLA + number of events / total events, where total events = number of murine CD45 + cells + CD33 + HLA + number of events.

  ** Naive samples were stained on 3 separate days of 3 separate studies. The value reflects the detection limit of the assay because mice were not injected with MV4-11 tumor cells.

  *** Statistical significance is unpaired two-tailed Student t test with Welch correction to account for unequal variance in the data, even for events where the sample size is too small for the Bartlett test for homogeneous variance Was used to determine.

  These data were 96% and 59% reduction in total body tumor mass in BM treated with VII when compared to the vehicle group (5 million and 10 million cell injection groups, respectively), and compared to the vehicle group Sometimes it shows 99% and 94% reduction in PB (5 million and 10 million cells, respectively) in tumor cell implantation with VII treatment (Table 4).

（）内の数字は、汚染が疑われる試料を除外するために調節された値を反映する（各１千万細胞ＰＢグループにおいてｎ＝１）。これらの試料は、心臓穿刺によって血液収集を試みている間、乳白色の浸出液を有することが注目され、その後、検死の際に、心臓または胸腺の近くに腫瘍を有することが注目された。これら２つの疑われた値は、ビヒクルおよび４０ｍｇ／ｋｇ ＶＩＩグループ（１千万細胞）においてそれぞれ９８％および９３％腫瘍細胞を示した。

Numbers in parentheses reflect values adjusted to exclude samples suspected of contamination (n = 1 in each 10 million cell PB group). These samples were noted to have a milky exudate while attempting to collect blood by cardiac puncture, and then at the time of necropsy to have a tumor near the heart or thymus. These two suspected values showed 98% and 93% tumor cells in the vehicle and 40 mg / kg VII group (10 million cells), respectively.

  Compound VII Pharmacokinetics in NOD-SCID Mice: Early PK data in NOD-SCID mice was generated in an early study prior to dosing in this study. Results show that dose proportional increase in plasma levels of VI at doses of 20, 40, or 80 mg / kg and exposure is limited to 2-4 hours after dosing, and plasma levels of VI are up to 6 hours after dosing in all female mice Indicates that it was virtually undetectable. Males showed higher exposure at comparable doses and longer periods of VI levels in plasma when compared to females.

  Additional PK data was obtained using the remaining dosing solution after the study was completed, due to precipitates observed in the formulation under investigation prepared in the initial study and other studies. NOD-SCID female mice (n = 4) were dosed with a 40 mg / kg dose solution of VII and plasma was obtained 1 hour after dosing. As shown in Table 5, the PK data with the residual formulation was moderately variable between the 4 animals with a percent coefficient of variation (% CV) value of 25%. The plasma VI concentrations obtained for 1 hour exposure were similar to those obtained in previous PK studies, indicating that these formulations were comparable.

  Tables 5A and 5B: PO administration of VII calcium salt: mean concentration of compound VI in plasma (ng / mL)

７．３．３ 結論
マウス匹あたり１千万細胞でのＭＶ４−１１腫瘍細胞の静脈内投与は、５百万細胞と比較して、多重の解剖学的部位への腫瘍のより大きい播種を結果として生じた。さらに、１千万細胞が接種されたマウスはより一貫して、骨髄および末梢血における植付けの高いレベルを有していた。播種されたＭＶ４−１１腫瘍を保有するマウスへの４０ｍｇ／ｋｇ ＶＩＩの投与は、細胞滴定とは関係なく、全体的生存率および病気関連後遺症に関して、ビヒクル処理マウスと比較したとき、顕著な有効性を証明した。加えて、ＶＩＩは、この調査において、６０日超の期間、ＮＯＤ−ＳＣＩＤマウスによって十分に許容され、用いられた配合物は、以前のＰＫ調査においてと同様な全身暴露を得るのに適しているように見えた。

7.3.3 Conclusion Intravenous administration of MV4-11 tumor cells at 10 million cells per mouse resulted in greater dissemination of the tumor to multiple anatomical sites compared to 5 million cells As a result. Furthermore, mice inoculated with 10 million cells had more consistent levels of implantation in bone marrow and peripheral blood. Administration of 40 mg / kg VII to seeded MV4-11 tumor-bearing mice is significantly more effective when compared to vehicle-treated mice with respect to overall survival and disease-related sequelae, regardless of cell titration Proved. In addition, VII is well tolerated by NOD-SCID mice in this study for over 60 days, and the formulation used is suitable for obtaining similar systemic exposure as in previous PK studies. It looked like.

  In summary, VII-treated mice have a total of tumors identified, the total number of tumor-bearing animals, and disease as measured by weight loss, autopsy status, and other live clinical observations. Proven reproducible drug-related reduction in severity. Data from this study were similar to previous efficacy studies, and treatment with VII showed repeated prolongation of survival in mice bearing MV4-11. In addition, mice treated with VII demonstrated fewer detectable MV4-11 tumor cells in bone marrow and peripheral blood when compared to vehicle controls. These data indicate that Syk / Flt-3 kinase inhibitors may be valuable therapeutics for the treatment of leukemia patients carrying FLT-3ITD or other leukemias associated with Flt-3 mutations. Suggests.

7.4 Example 4: Inhibition of constitutive Flt-3 activity in lysates from MV4-11 tumor grafts Initial investigations showed that treatment of MV4-11 human AML cells resulted in constitutive phosphorylation of Flt-3 It was shown that the inhibition occurred. Given these data, phosphorylation of Flt-3 relative to total Flt-3, tumor cell lysis from randomly selected mice from this study using human Flt-3 immunoprecipitation (IP) The product was evaluated ex vivo and then Western blot analysis of phosphorylated Flt-3 was performed as follows.

7.4.1 Experimental protocol Preparation of tumor lysate Collect tumors as soon as possible.
2. The frozen tumor is pulverized in liquid nitrogen.
3. Weigh and standardize in 250 mg / 1 ml ice cold RIPA buffer (Santa Cruz Biotechnology, sc-24948) containing protease inhibitor and phosphotase inhibitor cocktail (Sigma, P5726, 1 ml / 100 ml lysis buffer). .
4). Homogenize with ice.
The clear supernatant that is the tumor lysate is microcentrifuged and collected.

BCA Assay for Measuring Protein Concentration A BCA assay kit was obtained from Pierce Prod # 23227.
1. Preparation of BSA standard:
2000 ug / ml → 1500 ug / ml → 1000 ug / ml → 750 ug / ml → 500 ug / ml → 250 ug / ml → 125 ug / ml → 25 ug / ml
2. Predilut test samples with cold PBS (1:20 dilution of tumor lysate)
3. Incubate 200 ul BCA working solution + 25 ul standard / sample at 37 ° C. for 30 minutes.
4). Cool plate and read OD570nm.

Immunoprecipitation 1.1 mg tumor lysate is taken, 2 ug of primary antibody (anti-Flt-3 S-18, Santa Cruz, # sc-480) is added and the solution is adjusted to 500 ul with lysis buffer. Incubate with gentle shaking for 1 hour at 4 ° C.
2. Add 40ul of protein A / G plus-agarose beads (Santa Cruz, SC-2003). Incubate with gentle shaking for 1 hour at room temperature.
3. Microcentrifuge for 30 seconds at 4 ° C. Wash the pellet 3 times with 500 ul of 1 × lysis buffer. Keep on ice during washing.
4. Resuspend the pellet with 30ul of 2X Tris-Glycine SDS loading buffer + 5ul of reducing agent 10X (Invitrogen).
5. Heat the sample to 95-100 ° C. for 5 minutes.
6). Samples are stored at -80 ° C.

Western Blot Procedure Load 20ul of sample onto 1.8% Tris-Glycine gel.
Work in 2.1X Tris-Glycine SDS working buffer.
3. Transfer to PVDF membrane in 1 × Tris-Glycine transfer buffer.
4). Block in 1% BSA / TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature.
5. Primary antibody 1: 5,000 is added overnight at 4 ° C.

Anti-phosphotyrosine clone 4G10 (mouse monoclonal, Upstate, # 05-321).
Anti-phospho-Flt-3 (Tyr591) (mouse mAb, Cell Signaling, # 3466S).
6). Wash for 2 hours in TBST.
2 ° Ab anti-mouse IgG-HRP (Amersham, NA931V) 1: 5 for 1 hour in 7.5% milk / TBST (blotting grade blocking agent, non-fat dry milk, Bio-Rad, # 170-6404) , 000.
Brief exposure of ECL-plus (Amersham, RPN2132).

Membrane stripping and reprobing The membrane is submerged in stripping buffer (Pierce, # 21062) and incubated at 37 ° C. for 1 hour.
2. The membrane is washed twice for 15 minutes in TBST.
3. The membrane is blocked with 1% BSA / TBST for 1 hour.
Add anti-Flt-3 1: 5,000 (Santa Cruz, # sc-480) in 4.5% milk / TBST and incubate overnight at 4 ° C.
5. Wash for 2 hours in TBST.
6). 2 ° Ab anti-rabbit IgG-HRP (Amersham, NA934V) 1: 5,000 in 5% milk / TBST for 1 hour at room temperature.
7). Short-term exposure to ECL-Plus.

  In addition, the blot was reprobed for total Flt-3 levels. Constitutive phosphorylation of Flt-3 was reduced in tumors from 40 mg / kg VII treated animals in a dose-dependent manner when compared to vehicle treated controls.

7.4.2 Phosphorylated histone H3 analysis of MV4-11 tumor xenografts 3 from this study using immunohistochemical staining of human specific phosphorylated histone H3 (phH3) as a marker for tumor cell proliferation Growth was assessed ex vivo on formalin-fixed tumor sections from one randomly selected mouse. Human phH3 expression was reduced in tumor sections in a dose-dependent manner after treatment with VII. When compared to MV4-11 tumor xenografts from vehicle treated mice, treatment with 20 and 40 mg / kg VII resulted in 53% and 71% inhibition of phH3 staining, respectively. These data indicate that VII inhibition reduces the ability of MV4-11 tumors to grow in vivo and correlates with the reduced tumor volume observed during the survival portion of this study. Reduced proliferation may be due to decreased constitutive Flt-3 phosphorylation because this activity has been demonstrated to be required for MV4-11 cell survival in vitro. .

7.4.3 Inhibition of downstream signaling events Additional immunohistochemical staining of tumor sections from mice treated with 40 mg / kg VII for 26 days demonstrated reduced STAT5 and ERK1 / 2 phosphorylation. These are downstream molecules in the Flt-3: ITD signaling pathway in MV4-11 cells (FIG. 11).

7.5 Example 5: Investigation of Compound VI against Acute Myeloid Leukemia Cells and Comparison with Kinase Inhibitor AG1296 AML cells or 32D transfectants at 48 h (AML) or 24 h (32D) with 1 uM VI Treated and the% increase in apoptotic cells was determined using Annexin V and PI staining. The data shows that AML cells with Flt-3 ITD are sensitive only to VI-induced apoptosis. Similarly, 32D or point mutation Flt-3 TDK transfected with ITD undergoes more apoptosis at 24 hours than 32D cells transfected with wt Flt-3.

  The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. . These embodiments best illustrate the principles of the invention and its practical application, so that others skilled in the art will appreciate the various modifications with various modifications suitable for the invention and the particular use discussed. The embodiments have been selected and described to allow best use of the embodiments. The scope of the present invention is to be defined by the appended claims and their equivalents.

  All patents, patent applications, publications, and references cited herein are clearly intended to indicate that each individual publication or patent application is specifically and individually incorporated by reference. As such, they are incorporated by reference to the same extent.

Syk inhibitor compound VI demonstrates the ability to prevent proliferation and induce differentiation of TEL-Syk-transformed cells. DMSO, Syk- inhibitor compound VI (2 [mu] M), or Ab l kinase inhibitor either at 36 hours treated TEL-Syk- or BCRAb l of STI-571 (2μM) - DNA content of the transformed cells is shown ing. Syk inhibitor compound VI demonstrates the ability to prevent proliferation and induce differentiation of TEL-Syk-transformed cells. The ability of TEL-Syk to prevent pre-B cell differentiation has been shown. Syk inhibitor compound VI demonstrates the ability to prevent proliferation and induce differentiation of TEL-Syk-transformed cells. Cells were cultured for 3 days in the absence of IL-7 and analyzed for κLC expression by FACS. Inhibition of TEL-Syk activity with Compound VI has been shown. Syk-inhibitor VI has demonstrated the ability to prevent the growth of Myc-transformed pre-B cells. FIG. 2A is a FACS profile of pre-B cells transduced with Myc expression vector and cultured for 1 week in the absence of IL-7. Syk-inhibitor VI has demonstrated the ability to prevent the growth of Myc-transformed pre-B cells. The lower diagram of FIG. 2B shows the spleen of RAG / γC− ^+/− mice 5 weeks after the indicated cell infusion, illustrating the ability of Myc− transformed cells to cause splenomegaly and leukemia. FIG. 2B shows the DNA content of Myc-transformed cells treated for 36 hours with either DMSO, VI (2 μM), or STI-571 (2 μM). Syk-inhibitor VI has demonstrated the ability to prevent the growth of oncogenic SLP-65 ^{− / −} pre-B cell lines. FIG. 3A shows that splenomegaly and SLP-65 ^{− / −} pre-B cell lines are demonstrated by the spleen status of RAG / γC ^{− / −} mice 5 weeks after the indicated cell infusion. It shows that it can cause leukemia. Syk-inhibitor VI has demonstrated the ability to prevent the growth of oncogenic SLP-65 ^{− / −} pre-B cell lines. FIG. 3B shows the DNA content of tumorigenic SLP-65 ^{− / −} pre-B cell lines treated with either DMSO, VI (2 μM), or STI-571 (2 μM) for 36 hours. MV4-11 human acute myeloid leukemia cells were inoculated intravenously (iv) and treated with Compound VII at 40 mg / kg PO twice a day throughout the study from day 17 after tumor cell inoculation. Figure 2 shows the survival curve for the treated NOD-SCID mice. Calculation for ILS% of 40 mg / kg VII groups (10 ⁶ cells) with 50% of survival in this group at study termination (day 83), is assumed to median day of death of 83 days. The number of days until death for each animal is shown. Survival data are for NOD-SCID mice infused intravenously with MV4-11 tumor cells. Mice were treated twice daily with PO, vehicle or various doses of VII from day 17 after tumor cell injection to the end of the study (day 83). The number of days until death for the sacrificed animals is indicated in the graph as the median death date, indicated by the blue line. Surviving animals in each group at the end of the study are shown as surviving animals / total mice at the top of the graph. The tumor frequency in NOD-SCID mice bearing MV4-11 tumors at necropsy is shown. The severity of the disease was assessed by quantifying the frequency of mice with palpable tumors at the time of termination autopsy, regardless of the reason for sacrifice. The tumor frequency in NOD-SCID mice bearing MV4-11 tumors at necropsy is shown. The total number of tumors per group is shown in FIG. 6B. Mice were injected i.m. with 5 million or 10 million MV4-11 human AML cells (shown as 5E6 or 10E6 respectively). v. Vaccinated. On day 17 after cell injection, mice were treated with vehicle or 40 mg / kg VII (PO.bid) until sacrificed or throughout the study until day 83 for surviving animals. Mice were sacrificed and a comprehensive autopsy was performed. The total number of necropsy performed per group was 8-13. 2 shows the tumor distribution profile of experimental animals. The percentage of total tumors found at various anatomical locations was recorded at the time of sacrifice at necropsy. Mice received 5 million or 10 million MV4-11 cells i. v. Infused and dosed orally either vehicle or 40 mg / kg VII twice a day throughout the study. No palpable tumor was detected, but the graph includes animals that show expanded salivary glands. The total number of necropsy performed per group was 8-13. Figure 2 shows the MV4-11 tumor cell implantation profile in bone marrow (BM) and peripheral blood (PB) of NOD-SCID mice. Data are derived from tumor cells i. v. From the inoculation of MV4-11 human tumor cells in NOD-SCID mice treated with vehicle or 40 mg / kg VII twice daily PO, from day 17 after injection to the end of the study. Bone marrow (BM) and peripheral blood (PB) tumor cell implantation was detected by flow cytometric analysis using CD33 and HLA cell surface staining for detection of MV4-11 tumor cells. Data was normalized and percent human tumor cells from total cells were calculated. Total cells were defined as the number of human CD33 + HLA + positive events and murine CD45 positive staining events. The graph includes all samples. FIG. 5 shows detection of Flt-3 in MV4-11 xenografts by immunoprecipitation and Western blot analysis. Figure A shows MV4-11 tumor lysis from mice approximately 2 hours after the last dose of VII or vehicle using an anti-phosphotyrosine antibody on the left side of the blot and an anti-phospho-Flt-3 specific antibody on the right side of the blot. Is detection of phosphorylated Flt-3 in the product. In addition, blot reprobes for total Flt-3 levels are shown in FIG. FIG. 6 shows phosphorylated histone H3 analysis of MV4-11 tumor xenografts. Proliferation was observed in formalin-fixed tumor sections from 3 mice randomly selected from this study, using immunohistochemical staining of human specific phosphorylated histone H3 (phH3) as a marker for tumor cell growth. Rated ex vivo. Human phH3 expression was reduced in a dose-dependent manner in tumor sections after treatment with VII. When compared to MV4-11 tumor xenografts from vehicle treated mice, treatment with 20 and 40 mg / kg VII resulted in 53% and 71% inhibition of phH3 staining, respectively. These data indicate that VII-mediated inhibition reduced the ability of MV4-11 tumors to grow in vivo, which correlates with the reduction in tumor volume observed during the surviving portion of this study. Show. Reduced proliferation may be due to decreased constitutive Flt-3 phosphorylation, as it has been demonstrated that this activity is required for MV4-11 cell survival in vitro. It is. Shown are representative data illustrating immunohistochemical staining for pErk1 / 2 and pStat5 in tumor sections from MV4-11 tumor-bearing mice treated orally twice daily for 26 days. Tumors were collected from mice approximately 2 hours after the last dose of vehicle or 40 mg / kg VII. FIG. 5 shows the effect of treatment of AML cells or 32D transfectants with 1 uM VI on the induction of apoptosis as determined by Annexin V and PI staining.

Claims (25)

For the treatment of a cell proliferative disorder, a composition comprising a Syk kinase inhibitory compound, the compound, (N4-(2,2-dimethyl-4 - [(dihydrogen phosphonoxy) methyl] -3-oxo -5-pyrido [1,4] oxazin-6-yl) -5-fluoro-N2- (3,4,5-trimethoxyphenyl) -2,4-pyrimidinediamine)

Or a salt, hydrate, solvate, or N-oxide thereof. The composition of claim 1, wherein the cell proliferative disorder is a hematopoietic neoplasm. It said hematopoietic neoplasm, Ru neoplasms der lymphoid composition of claim 2. 4. The composition of claim 3, wherein the lymphoid neoplasm is a T cell neoplasm. 5. The composition of claim 4, wherein the T cell neoplasm is T lymphoblastic leukemia. 4. The composition of claim 3, wherein the lymphoid neoplasm is a B cell neoplasm. 7. The composition of claim 6, wherein the B cell neoplasm is B-lymphoblastic leukemia or Burkitt lymphoma. The composition of claim 1, wherein the cell proliferative disorder is a bone marrow neoplasm. The bone marrow neoplasm is
(I) or Ru der myeloproliferative disorders;
(Ii) or Ru der myelodysplastic disorders;
(Iii) either a myelodysplastic syndrome, or (iv) Ru Der acute myeloid leukemia, the composition of claim 8. 10. The composition of claim 9, wherein the myeloproliferative disorder is chronic myelogenous leukemia (CML). 10. The composition of claim 9, wherein the myelodysplastic disease is chronic myelomonocytic leukemia. 10. The composition of claim 9, wherein the acute myeloid leukemia is associated with the activity of a TEL / Syk fusion protein. The hematopoietic neoplasm is selected from acute myeloid leukemia, B-progenitor cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, myelodysplastic syndrome, and chronic myelogenous leukemia. 2. The composition according to 2. 2. The composition of claim 1, wherein the proliferative disorder is a virus mediated tumor resulting from modulation of Syk kinase activity. (I) the virally mediated tumor is either associated with virus encoding ITAM motif; or (ii) the virally mediated tumor is not associated with Kaposi's sarcoma-associated herpesvirus Luke; or (iii) the virus mediated tumor, Epstein - Luke was associated with Barr virus; or the virally mediated tumor is associated with HTLV-1 virus, the composition according to claim 14. 16. The composition of claim 15, wherein the virus-mediated tumor is associated with the activity of the K1 protein of Kaposi's sarcoma-associated herpesvirus. 16. The composition of claim 15, wherein the virus-mediated tumor is associated with activity of the Epstein-Barr virus LMP2A protein. The composition of claim 1, wherein the composition comprises a mixture of Syk inhibitor compounds. It said composition, you comprising administering additionally with Abl kinase inhibitor composition of claim 1. (I) the Abl kinase inhibitor is 2-phenylaminopyrimidine; or
20. The composition of claim 19, wherein (ii) the composition and the Abl kinase inhibitor are administered sequentially or simultaneously. To inhibit tumor metastasis, a composition comprising a Syk kinase inhibitory compound, the compound, (N4-(2,2-dimethyl-4 - [(dihydrogen phosphonoxy) methyl] -3-oxo -5 -Pyrid [1,4] oxazin-6-yl) -5-fluoro-N2- (3,4,5-trimethoxyphenyl) -2,4-pyrimidinediamine)

Or a salt, hydrate, solvate, or N-oxide thereof. Wherein the tumor is breast cancer, ovarian cancer, renal cancer, gastrointestinal cancer, renal cancer, bladder cancer, pancreatic cancer, Ru is selected from lung squamous carcinoma, and adenocarcinoma, A composition according to claim 2 1. 23. The composition of claim 22, wherein the composition is administered prophylactically prior to diagnosis of tumor metastasis. The metastasis, Ru caused by integrin signaling through Syk, composition of claim 23. 25. The composition of claim 24, wherein the transfer is caused by integrin signaling by β1 integrin, β2 integrin, or β3 integrin.

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